Rapid quantitative analysis of proteins or protein function in complex mixtures

ABSTRACT

Analytical reagents and mass spectrometry-based methods using these reagents for the rapid, and quantitative analysis of proteins or protein function in mixtures of proteins. The methods employ affinity labeled protein reactive reagents having three portions: an affinity label (A) covalently linked to a protein reactive group (PRG) through a linker group (L). The linker may be differentially isotopically labeled, e.g., by substitution of one or more atoms in the linker with a stable isotope thereof. These reagents allow for the selective isolation of peptide fragments or the products of reaction with a given protein (e.g., products of enzymatic reaction) from complex mixtures. The isolated peptide fragments or reaction products are characteristic of the presence of a protein or the presence of a protein function in those mixtures. Isolated peptides or reaction products are characterized by mass spectrometric (MS) techniques. The reagents also provide for differential isotopic labeling of the isolated peptides or reaction products which facilitates quantitative determination by mass spectrometry of the relative amounts of proteins in different samples. The methods of this invention can be used for qualitative and quantitative analysis of global protein expression profiles in cells and tissues, to screen for and identify proteins whose expression level in cells, tissue or biological fluids is affected by a stimulus or by a change in condition or state of the cell, tissue or organism from which the sample originated.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of U.S. patent application Ser.No. 09/388,062, filed Aug. 25, 1999, which takes priority under 35U.S.C.§119(e) from U.S. provisional application serial No. 60/097,788,filed Aug. 25, 1998, and serial No. 60/099,113, filed Sep. 3, 1998, allof which are incorporated in their entirety by reference herein.

[0002] This invention was made through funding from the National ScienceFoundation Science and Technology Center for Molecular Biotechnology(grants 5T32HG and BIR9214821) and the National Institutes of Health(NIH grants RR11823, T32HG00035, HD-02274 and GM60184). The UnitedStates government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Genomic technology has advanced to a point at which, inprinciple, it has become possible to determine complete genomicsequences and to quantitatively measure the mRNA levels for each geneexpressed in a cell. For some species the complete genomic sequence hasnow been determined, and for one strain of the yeast Saccharomycescervisiae, the mRNA levels for each expressed gene have been preciselyquantified under different growth conditions (Velculescu et al., 1997).Comparative cDNA array analysis and related technologies have been usedto determine induced changes in gene expression at the mRNA level byconcurrently monitoring the expression level of a large number of genes(in some cases all the genes) expressed by the investigated cell ortissue (Shalon et al., 1996). Furthermore, biological and computationaltechniques have been used to correlate specific function with genesequences. The interpretation of the data obtained by these techniquesin the context of the structure, control and mechanism of biologicalsystems has been recognized as a considerable challenge. In particular,it has been extremely difficult to explain the mechanism of biologicalprocesses by genomic analysis alone.

[0004] Proteins are essential for the control and execution of virtuallyevery biological process. The rate of synthesis and the half-life ofproteins and thus their expression level are also controlledpost-transcriptionally. Furthermore, the activity of proteins isfrequently modulated by post-translational modifications, in particularprotein phosphorylation, and dependent on the association of the proteinwith other molecules including DNA and proteins. Neither the level ofexpression nor the state of activity of proteins is therefore directlyapparent from the gene sequence or even the expression level of thecorresponding mRNA transcript. It is therefore essential that a completedescription of a biological system include measurements that indicatethe identity, quantity and the state of activity of the proteins whichconstitute the system. The large-scale (ultimately global) analysis ofproteins expressed in a cell or tissue has been termed proteome analysis(Pennington et al., 1997).

[0005] At present no protein analytical technology approaches thethroughput and level of automation of genomic technology. The mostcommon implementation of proteome analysis is based on the separation ofcomplex protein samples most commonly by two-dimensional gelelectrophoresis (2DE) and the subsequent sequential identification ofthe separated protein species (Ducret et al., 1998; Garrels et al.,1997; Link et al., 1997; Shevchenko et al., 1996; Gygi et al. 1999;Boucherie et al., 1996). This approach has been revolutionized by thedevelopment of powerful mass spectrometric techniques and thedevelopment of computer algorithms which correlate protein and peptidemass spectral data with sequence databases and thus rapidly andconclusively identify proteins (Eng et al., 1994; Mann and Wilm, 1994;Yates et al., 1995). This technology has reached a level of sensitivitywhich now permits the identification of essentially any protein which isdetectable by conventional protein staining methods including silverstaining (Figeys and Aebersold, 1998; Figeys et al., 1996; Figeys etal., 1997; Shevchenko et al., 1996). However, the sequential manner inwhich samples are processed limits the sample throughput, the mostsensitive methods have been difficult to automate and low abundanceproteins, such as regulatory proteins, escape detection without priorenrichment, thus effectively limiting the dynamic range of thetechnique. In the 2DE/(MS)N method, proteins are quantified bydensitometry of stained spots in the 2DE gels.

[0006] The development of methods and instrumentation for automated,data-dependent electrospray ionization (ESI) tandem mass spectrometry(MS^(n)) in conjunction with microcapillary liquid chromatography (μLC)and database searching has significantly increased the sensitivity andspeed of the identification of gel-separated proteins. As an alternativeto the 2DE/MS^(n) approach to proteome analysis, the direct analysis bytandem mass spectrometry of peptide mixtures generated by the digestionof complex protein mixtures has been proposed (Dongr'e et al., 1997).μLC-Ms/MS has also been used successfully for the large-scaleidentification of individual proteins directly from mixtures without gelelectrophoretic separation (Link et al., 1999; Opitek et al., 1997)While these approaches dramatically accelerate protein identification,the quantities of the analyzed proteins cannot be easily determined, andthese methods have not been shown to substantially alleviate the dynamicrange problem also encountered by the 2DE/MS/MS approach. Therefore, lowabundance proteins in complex samples are also difficult to analyze bythe uLC/MS/MS method without their prior enrichment.

[0007] It is therefore apparent that current technologies, whilesuitable to identify the components of protein mixtures, are neithercapable of measuring the quantity nor the state of activity of theprotein in a mixture. Even evolutionary improvements of the currentapproaches are unlikely to advance their performance sufficiently tomake routine quantitative and functional proteome analysis a reality.

[0008] This invention provides methods and reagents that can be employedin proteome analysis which overcome the limitations inherent intraditional techniques. The basic approach described can be employed forthe quantitative analysis of protein expression in complex samples (suchas cells, tissues, and fractions thereof), the detection andquantitation of specific proteins in complex samples, and thequantitative measurement of specific enzymatic activities in complexsamples.

[0009] In this regard, a multitude of analytical techniques arepresently available for clinical and diagnostic assays which detect thepresence, absence, deficiency or excess of a protein or protein functionassociable with a normal or disease state. While these techniques arequite sensitive, they do not necessarily provide chemical speciation ofproducts and may, as a result, be difficult to use for assaying severalproteins or enzymes simultaneously in a single sample. Current methodsmay not distinguish among aberrant expression of different enzymes ortheir malfunctions which lead to a common set of clinical symptoms. Themethods and reagents herein can be employed in clinical and diagnosticassays for simultaneous (multiplex) monitoring of multiple proteins andprotein reactions.

SUMMARY OF THE INVENTION

[0010] This invention provides analytical reagents and massspectrometry-based methods using these reagents for the rapid, andquantitative analysis of proteins or protein function in mixtures ofproteins. The analytical method can be used for qualitative andparticularly for quantitative analysis of global protein expressionprofiles in cells and tissues, i.e. the quantitative analysis ofproteomes. The method can also be employed to screen for and identifyproteins whose expression level in cells, tissue or biological fluids isaffected by a stimulus (e.g., administration of a drug or contact with apotentially toxic material), by a change in environment (e.g., nutrientlevel, temperature, passage of time) or by a change in condition or cellstate (e.g., disease state, malignancy, site-directed mutation, geneknockouts) of the cell, tissue or organism from which the sampleoriginated. The proteins identified in such a screen can function asmarkers for the changed state. For example, comparisons of proteinexpression profiles of normal and malignant cells can result in theidentification of proteins whose presence or absence is characteristicand diagnostic of the malignancy.

[0011] In an exemplary embodiment, the methods herein can be employed toscreen for changes in the expression or state of enzymatic activity ofspecific proteins. These changes may be induced by a variety ofchemicals, including pharmaceutical agonists or antagonists, orpotentially harmful or toxic materials. The knowledge of such changesmay be useful for diagnosing enzyme-based diseases and for investigatingcomplex regulatory networks in cells.

[0012] The methods herein can also be used to implement a variety ofclinical and diagnostic analyses to detect the presence, absence,deficiency or excess of a given protein or protein function in abiological fluid (e.g., blood), or in cells or tissue. The method isparticularly useful in the analysis of complex mixtures of proteins,i.e., those containing 5 or more distinct proteins or protein functions.

[0013] The inventive method employs affinity-labeled protein reactivereagents that allow for the selective isolation of peptide fragments orthe products of reaction with a given protein (e.g., products ofenzymatic reaction) from complex mixtures. The isolated peptidefragments or reaction products are characteristic of the presence of aprotein or the presence of a protein function, e.g., an enzymaticactivity, respectively, in those mixtures. Isolated peptides or reactionproducts are characterized by mass spectrometric (MS) techniques. Inparticular, the sequence of isolated peptides can be determined usingtandem MS (MS^(n)) techniques, and by application of sequence databasesearching techniques, the protein from which the sequenced peptideoriginated can be identified. The reagents also provide for differentialisotopic labeling of the isolated peptides or reaction products whichfacilitates quantitative determination by mass spectrometry of therelative amounts of proteins in different samples. Also, the use ofdifferentially isotopically-labeled reagents as internal standardsfacilitates quantitative determination of the absolute amounts of one ormore proteins or reaction products present in the sample.

[0014] In general, the affinity labeled protein reactive reagents ofthis invention have three portions: an affinity label (A) covalentlylinked to a protein reactive group (PRG) through a linker group (L):

A—L—PRG

[0015] The linker may be differentially isotopically labeled, e.g., bysubstitution of one or more atoms in the linker with a stable isotopethereof. For example, hydrogens can be substituted with deuteriums orC¹² with C¹³.

[0016] The affinity label A functions as a molecular handle thatselectively binds covalently or non-covalently, to a capture reagent(CR). Binding to CR facilitates isolation of peptides, substrates orreaction products tagged or labeled with A. In specific embodiments, Ais a strepavidin or avidinn. After affinity isolation of affinity taggedmaterials, some of which may be isotopically labeled, the interactionbetween A and the capture reagent is disrupted or broken to allow MSanalysis of the isolated materials. The affinity label may be displacedfrom the capture reagent by addition of displacing ligand, which may befree A or a derivative of A, or by changing solvent (e.g., solvent typeor pH) or temperature conditions or the linker may be cleavedchemically, enzymatically, thermally or photochemically to release theisolated materials for MS analysis.

[0017] Two types of PRG groups are specifically provided herein: (a)those groups that selectively react with a protein functional group toform a covalent or non-covalent bond tagging the protein at specificsites, and (b) those that are transformed by action of the protein,e.g., that are substrates for an enzyme. In specific embodiments, PRG isa group having specific reactivity for certain protein groups, such asspecificity for sulfhydryl groups, and is useful in general forselectively tagging proteins in complex mixtures. A sulfhydryl specificreagent tags proteins containing cysteine. In other specificembodiments, PRG is an enzyme substrate that is selectively cleaved(leaving A—L) or modified (giving A—L—PRG′) by the action of an enzymeof interest.

[0018] Exemplary reagents have the general formula:

A—B¹—X¹—(CH₂)_(n)—[X²—(CH₂)_(m)]_(x)—X³—(CH₂)_(p)—X⁴—B²—PRG

[0019] where:

[0020] A is the affinity label;

[0021] PRG is the protein reactive group;

[0022] X¹, X², X³ and X⁴, independently of one another, and X²independently of other X² in the linker group, can be selected from O,S, NH, NR, NRR′⁺, CO, COO, COS, S—S, SO, SO₂, CO—NR′, CS—NR′, Si—O, arylor diaryl groups or X¹—X⁴ may be absent, but preferably at least one ofX¹—X⁴ is present;

[0023] B¹ and B², independently of one another, are optional moietiesthat can faciliate bonding of the A or PRG group to the linker orprevent undesired cleavage of those groups from the linker and can beselected, for example, from COO, CO, CO—NR′, CS—NR′ and may contain oneor more CH₂ groups alone or in combination with other groups,e.g.(CH₂)_(q)—CONR′, (CH₂)_(q)—CS—NR′, or (CH₂)_(q);

[0024] n, m, p and q are whole numbers that can have values from 0 toabout 100, preferably one of n, m, p or q is not 0 and x is also a wholenumber that can range from 0 to about 100 where the sum of n+xm+p+q ispreferably less than about 100 and more preferably less than about 20;

[0025] R is an alkyl, alkenyl, alkynyl, alkoxy or aryl group; and

[0026] R′ is a hydrogen, an alkyl, alkenyl, alkynyl, alkoxy or arylgroup.

[0027] One or more of the CH₂ groups of the linker can be optionallysubstituted with small (C1-C6) alkyl, alkenyl, or alkoxy groups, an arylgroup or can be substituted with functional groups that promoteionization, such as acidic or basic groups or groups carrying permanentpositive or negative charge. One or more single bonds connecting CH₂groups in the linker can be replaced with a double or a triple bond.Preferred R and R′ alkyl, alkenyl, alkynyl or alkoxy groups are smallhaving 1 to about 6 carbon atoms.

[0028] One or more of the atoms in the linker can be substituted with astable isotope to generate one or more substantially chemicallyidentical, but isotopically distinguishable reagents. For example, oneor more hydrogens in the linker can be substituted with deuterium togenerate isotopically heavy reagents.

[0029] In an exemplary embodiment the linker contains groups that can becleaved to remove the affinity tag. If a cleavable linker group isemployed, it is typically cleaved after affinity tagged peptides,substrates or reaction products have been isolated using the affinitylabel together with the CR. In this case, any isotopic labeling in thelinker preferably remains bound to the protein, peptide, substrate orreaction product.

[0030] Linker groups include among others: ethers, polyethers, etherdiamines, polyether diamines, diamines, amides, polyamides,polythioethers, disulfides, silyl ethers, alkyl or alkenyl chains(straight chain or branched and portions of which may be cyclic), aryl,diaryl or alkyl-aryl groups. Aryl groups in linkers can contain one ormore heteroatoms (e.g., N, O or S atoms).

[0031] In one aspect, the invention provides a mass spectrometric methodfor identification and quantitation of one or more proteins in a complexmixture which employs affinity labeled reagents in which the PRG is agroup that selectively reacts with certain groups that are typicallyfound in peptides (e.g., sulfhydryl, amino, carboxy, homoserine lactonegroups). One or more affinity labeled reagents with different PRG groupsare introduced into a mixture containing proteins and the reagents reactwith certain proteins to tag them with the affinity label. It may benecessary to pretreat the protein mixture to reduce disulfide bonds orotherwise facilitate affinity labeling. After reaction with the affinitylabeled reagents, proteins in the complex mixture are cleaved, e.g.,enzymatically, into a number of peptides. This digestion step may not benecessary, if the proteins are relatively small. Peptides that remaintagged with the affinity label are isolated by an affinity isolationmethod, e.g., affinity chromatography, via their selective binding tothe CR. Isolated peptides are released from the CR by displacement of Aor cleavage of the linker, and released materials are analyzed by liquidchromatography/mass spectrometry (LC/MS). The sequence of one or moretagged peptides is then determined by MS^(n) techniques. At least onepeptide sequence derived from a protein will be characteristic of thatprotein and be indicative of its presence in the mixture. Thus, thesequences of the peptides typically provide sufficient information toidentify one or more proteins present in a mixture.

[0032] Quantitative relative amounts of proteins in one or moredifferent samples containing protein mixtures (e.g., biological fluids,cell or tissue lysates, etc.) can be determined using chemicallyidentical, affinity tagged and differentially isotopically labeledreagents to affinity tag and differentially isotopically label proteinsin the different samples. In this method, each sample to be compared istreated with a different isotopically labeled reagent to tag certainproteins therein with the affinity label. The treated samples are thencombined, preferably in equal amounts, and the proteins in the combinedsample are enzymatically digested, if necessary, to generate peptides.Some of the peptides are affinity tagged and in addition tagged peptidesoriginating from different samples are differentially isotopicallylabeled. As described above, affinity labeled peptides are isolated,released from the capture reagent and analyzed by (LC/MS). Peptidescharacteristic of their protein origin are sequenced using MS^(n)techniques allowing identification of proteins in the samples. Therelative amounts of a given protein in each sample is determined bycomparing relative abundance of the ions generated from anydifferentially labeled peptides originating from that protein. Themethod can be used to assess relative amounts of known proteins indifferent samples. Further, since the method does not require any priorknowledge of the type of proteins that may be present in the samples, itcan be used to identify proteins which are present at different levelsin the samples examined. More specifically, the method can be applied toscreen for and identify proteins which exhibit differential express incells, tissue or biological fluids. It is also possible to determine theabsolute amounts of specific proteins in a complex mixture. In thiscase, a known amount of internal standard, one for each specific proteinin the mixture to be quantified, is added to the sample to be analyzed.The internal standard is an affinity tagged peptide that is identical inchemical structure to the affinity tagged peptide to be quantifiedexcept that the internal standard is differentially isotopicallylabeled, either in the peptide or in the affinity tag portion, todistinguish it from the affinity tagged peptide to be quantified. Theinternal standard can be provided in the sample to be analyzed in otherways. For example, a specific protein or set of proteins can bechemically tagged with an isotopically-labeled affinity tagging reagent.A known amount of this material can be added to the sample to beanalyzed. Alternatively, a specific protein or set of proteins may belabeled with heavy atom isotopes and then derivatized with an affinitytagging reagent.

[0033] Also, it is possible to quantify the levels of specific proteinsin multiple samples in a single analysis (multiplexing). In this case,affinity tagging reagent s used to derivatize proteins present indifferent affinity tagged peptides from different samples can beselectively quantified by mass spectrometry.

[0034] In this aspect of the invention, the method provides forquantitative measurement of specific proteins in biological fluids,cells or tissues and can be applied to determine global proteinexpression profiles in different cells and tissues. The same generalstrategy can be broadened to achieve the proteome-wide, qualitative andquantitative analysis of the state of modification of proteins, byemploying affinity reagents with differing specificity for reaction withproteins. The method and reagents of this invention can be used toidentify low abundance proteins in complex mixtures and can be used toselectively analyze specific groups or classes of proteins such asmembrane or cell surface proteins, or proteins contained withinorganelles, sub-cellular fractions, or biochemical fractions such asimmunoprecipitates. Further, these methods can be applied to analyzedifferences in expressed proteins in different cell states. For example,the methods and reagents herein can be employed in diagnostic assays forthe detection of the presence or the absence of one or more proteinsindicative of a disease state, such as cancer.

[0035] In a second aspect, the invention provides a MS method fordetection of the presence or absence of a protein function, e.g., anenzyme activity, in a sample. The method can also be employed to detecta deficiency or excess (over normal levels) of protein function in asample. Samples that can be analyzed include various biological fluidsand materials, including tissue and cells. In this case, the PRG of theaffinity labeled reagent is a substrate for the enzyme of interest.Affinity labeled substrates are provided for each enzyme of interest andare introduced into a sample where they react to generate affinitylabeled products, if the enzyme of interest is present in the sample.Products or unreacted substrate that are tagged with the affinity labelare isolated by an affinity isolation method, e.g., affinitychromatography, via their selective binding to the CR. The isolatedtagged substrates and products are analyzed by mass spectrometry.Affinity labeled products include those in which the substrate isentirely cleaved from the linker or in which the substrate is modifiedby reaction with a protein of interest. Detection of theaffinity-labeled product indicates the protein function is present inthe sample. Detection of little or no affinity labeled product indicatesdeficiency or absence, respectively, of the protein function in thesample.

[0036] The amount of selected protein, e.g., measured in terms of enzymeactivity, present in a sample can be measured by introducing a knownamount of an internal standard which is an isotopically labeled analogof the expected product of the enzymatic reaction of the reagentsubstrate. The internal standard is substantially chemically identicalto the expected enzymatic reaction product, but is isotopicallydistinguishable therefrom. The level of protein function (e.g.,enzymatic activity) in a given sample can be compared with activitylevels in other samples or controls (either negative or positivecontrols). The procedure therefore can detect the presence, absence,deficiency or excess of a protein function in a sample. The method iscapable of quantifying the velocity of an enzymatic reaction since itenables the amount of product formed over a known time period to bemeasured. This method can be multiplexed, by simultaneous use of aplurality of affinity labeled substrates selective for different proteinfunctions and if quantitation is desired by inclusion of thecorresponding internal standards for expected products, to analyze for aplurality of protein functions in a single sample.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The methods of this invention employ affinity tagged proteinreactive reagents in which the affinity tag is covalently attached to aprotein reactive group by a linker. The linker can be isotopicallylabeled to generate pairs or sets of reagents that are substantiallychemically identical, but which are distinguishable by mass. For examplea pair of reagents, one of which is isotopically heavy and the other ofwhich is isotopically light can be employed for the comparison of twosamples one of which may be a reference sample containing one or moreknown proteins in known amounts. For example, any one or more of thehydrogen, nitrogen, oxygen or sulfur atoms in the linker may be replacedwith their isotopically stable isotopes: ²H, ¹³C, ¹⁵N, ¹⁷O, ¹⁸O or ³⁴S.

[0038] Suitable affinity tags bind selectively either covalently ornon-covalently and with high affinity to a capture reagent (CR). TheCR—A interaction or bond should remain intact after extensive andmultiple washings with a variety of solutions to remove non-specificallybound components. The affinity tag binds minimally or preferably not atall to components in the assay system, except CR, and does notsignificantly bind to surfaces of reaction vessels. Any non-specificinteraction of the affinity tag with other components or surfaces shouldbe disrupted by multiple washes that leave CR—A intact. Further, it mustbe possible to disrupt the interaction of A and CR to release peptides,substrates or reaction products, for example, by addition of adisplacing ligand or by changing the temperature or solvent conditions.Preferably, neither CR or A react chemically with other components inthe assay system and both groups should be chemically stable over thetime period of an assay or experiment. The affinity tag preferably doesnot undergo peptide-like fragmentation during (MS)^(n) analysis. Theaffinity label is preferably soluble in the sample liquid to be analyzedand the CR should remain soluble in the sample liquid even thoughattached to an insoluble resin such as Agarose. In the case of CR termsoluble means that CR is sufficiently hydrated or otherwise solvatedsuch that it functions properly for binding to A. CR or CR-containingconjugates should not be present in the sample to be analyzed, exceptwhen added to capture A.

[0039] Examples of A and CR Pairs Include:

[0040] d-biotin or structurally modified biotin-based reagents,including d-iminobiotin, which bind to proteins of theavidin/streptavidin, which may, for example, be used in the forms ofstrepavidin-Agarose, oligomeric-avidin-Agarose, ormonomeric-avidin-Agarose;

[0041] any 1,2-diol, such as 1,2-dihydroxyethane (HO—CH₂—CH₂—OH), andother 1,2-dihydroxyalkanes including those of cyclic alkanes, e.g.,1,2-dihydroxycyclohexane which bind to an alkyl or aryl boronic acid orboronic acid esters, such as phenyl-B(OH)₂ or hexyl-B(OEthyl)₂ which maybe attached via the alkyl or aryl group to a solid support material,such as Agarose;

[0042] maltose which binds to maltose binding protein (as well as anyother sugar/sugar binding protein pair or more generally to anyligand/ligand binding protein pairs that has properties discussedabove);

[0043] a hapten, such as dinitrophenyl group, for any antibody where thehapten binds to an anti-hapten antibody that recognizes the hapten, forexample the dinitrophenyl group will bind to an anti-dinitrophenyl-lgG;

[0044] a ligand which binds to a transition metal, for example, anoligomeric histidine will bind to Ni(II), the transition metal CR may beused in the form of a resin bound chelated transition metal, such asnitrilotriacetic acid-chelated Ni(II) or iminodiacetic acid-chelatedNi(II);

[0045] glutathione which binds to glutathione-S-transferase.

[0046] In general, any A—CR pair commonly used for affinity enrichmentwhich meets the suitability criteria discussed above. Biotin andbiotin-based affinity tags are preferred. Of particular interest arestructurally modified biotins, such as d-iminobiotin, which will elutefrom avidin or strepavidin columns under solvent conditions compatiblewith ESI-MS analysis, such as dilute acids containing 10-20% organicsolvent. It is expected that d-iminobiotin tagged compounds will elutein solvents below pH 4. d-Iminobiotin tagged protein reactive reagentscan be synthesized by methods described herein for the correspondingbiotin tagged reagents.

[0047] A displacement ligand, DL, is optionally used to displace A fromCR. Suitable DLs are not typically present in samples unless added. DLshould be chemically and enzymatically stable in the sample to beanalyzed and should not react with or bind to components (other than CR)in samples or bind non-specifically to reaction vessel walls. DLpreferably does not undergo peptide-like fragmentation during MSanalysis, and its presence in sample should not significantly suppressthe ionization of tagged peptide, substrate or reaction productconjugates.

[0048] DL itself preferably is minimally ionized during massspectrometric analysis and the formation of ions composed of DL clustersis preferably minimal. The selection of DL, depends upon the A and CRgroups that are employed. In general, DL is selected to displace A fromCR in a reasonable time scale, at most within a week of its addition,but more preferably within a few minutes or up to an hour. The affinityof DL for CR should be comparable or stronger than the affinity of thetagged compounds containing A for CR. Furthermore, DL should be solublein the solvent used during the elution of tagged compounds containing Afrom CR. DL preferably is free A or a derivative or structuralmodification of A. Examples of DL include, d-biotin or d-biotinderivatives, particularly those containing groups that suppress clusterformation or suppress ionization in MS.

[0049] The linker group (L) should be soluble in the sample liquid to beanalyzed and it should be stable with respect to chemical reaction,e.g., substantially chemically inert, with components of the sample aswell as A and CR groups. The linker when bound to A should not interferewith the specific interaction of A with CR or interfere with thedisplacement of A from CR by a displacing ligand or by a change intemperature or solvent. The linker should bind minimally or preferablynot at all to other components in the system, to reaction vesselsurfaces or CR. Any non-specific interactions of the linker should bebroken after multiple washes which leave the A—CR complex intact.Linkers preferably do not undergo peptide-like fragmentation during(MS)^(n) analysis. At least some of the atoms in the linker groupsshould be readily replaceable with stable heavy-atom isotopes. Thelinker preferably contains groups or moieties that facilitate ionizationof the affinity tagged reagents, peptides, substrates or reactionproducts.

[0050] To promote ionization, the linker may contain acidic or basicgroups, e.g., COOH, SO₃H, primary, secondary or tertiary amino groups,nitrogen-heterocycles, ethers, or combinations of these groups. Thelinker may also contain groups having a permanent charge, e.g.,phosphonium groups, quaternary ammonium groups, sulfonium groups,chelated metal ions, tetralky or tetraryl borate or stable carbanions.

[0051] The covalent bond of the linker to A or PRG should typically notbe unintentionally cleaved by chemical or enzymatic reactions during theassay. In some cases it may be desirable to cleave the linker from theaffinity tag A or from the PRG, for example to facilitate release froman affinity column. Thus, the linker can be cleavable, for example, bychemical, thermal or photochemical reaction. Photocleavable groups inthe linker may include the 1-(2-nitrophenyl)-ethyl group. Thermallylabile linkers may, for example, be a double-stranded duplex formed fromtwo complementary strands of nucleic acid, a strand of a nucleic acidwith a complementary strand of a peptide nucleic acid, or twocomplementary peptide nucleic acid strands which will dissociate uponheating. Cleavable linkers also include those having disulfide bonds,acid or base labile groups, including among others, diarylmethyl ortrimethylarylmethyl groups, silyl ethers, carbamates, oxyesters,thiesters, thionoesters, and α-fluorinated amides and esters.Enzymatically cleavable linkers can contain, for example,protease-sensitive amides or esters, β-lactamase-sensitive β-lactamanalogs and linkers that are nuclease-cleavable, orglycosidase-cleavable.

[0052] The protein reactive group (PRG) can be a group that selectivelyreacts with certain protein functional groups or is a substrate of anenzyme of interest. Any selectively reactive protein reactive groupshould react with a functional group of interest that is present in atleast a portion of the proteins in a sample. Reaction of PRG withfunctional groups on the protein should occur under conditions that donot lead to substantial degradation of the compounds in the sample to beanalyzed. Examples of selectively reactive PRGs suitable for use in theaffinity tagged reagents of this invention, include those which reactwith sulfhydryl groups to tag proteins containing cysteine, those thatreact with amino groups, carboxylate groups, ester groups, phosphatereactive groups, and aldehyde and/or ketone reactive groups or, afterfragmentation with CNBr, with homoserine lactone.

[0053] Thiol reactive groups include epoxides, α-haloacyl group,nitriles, sulfonated alkyl or aryl thiols and maleimides. Amino reactivegroups tag amino groups in proteins and include sulfonyl halides,isocyanates, isothiocyanantes, active esters, includingtetrafluorophenyl esters, and N-hydroxysuccinimidyl esters, acidhalides, and acid anyhydrides. In addition, amino reactive groupsinclude aldehydes or ketones in the presence or absence of NaBH₄ orNaCNBH_(3.)

[0054] Carboxylic acid reactive groups include amines or alcohols in thepresence of a coupling agent such as dicyclohexylcarbodiimide, or2,3,5,6-tetrafluorophenyl trifluoroacetate and in the presence orabsence of a coupling catalyst such as 4-dimethylaminopyridine; andtransition metal-diamine complexes including Cu(II)phenanthroline

[0055] Ester reactive groups include amines which, for example, reactwith homoserine lactone.

[0056] Phosphate reactive groups include chelated metal where the metalis, for example Fe(III) or Ga(III), chelated to, for example,nitrilotriacetiac acid or iminodiacetic acid.

[0057] Aldehyde or ketone reactive groups include amine plus NaBH₄ orNaCNBH₃, or these reagents after first treating a carbohydrate withperiodate to generate an aldehyde or ketone.

[0058] PRG groups can also be substrates for a selected enzyme ofinterest. The enzyme of interest may, for example, be one that isassociated with a disease state or birth defect or one that is routinelyassayed for medical purposes. Enzyme substrates of interest for use withthe methods of this invention include, acid phosphatase, alkalinephosphatase, alanine aminotransferase, amylase, angiotensin convertingenzyme, aspartate aminotransferase, creatine kinase,gamma-glutamyltransferase, lipase, lactate dehydrogenase, andglucose-6-phosphate dehydrogenase which are currently routinely assayedby other methods.

[0059] The requirements discussed above for A, L, PRG, extend to thecorresponding to the segments of A—L—PRG and the reaction productsgenerated with this reagent.

[0060] Internal standards, which are appropriately isotopically labeled,may be employed in the methods of this invention to measure absolutequantitative amounts of proteins in samples. Internal standards are ofparticular use in assays intended to quantitate affinity tagged productsof enzymatic reactions. In this application, the internal standard ischemically identical to the tagged enzymatic product generated by theaction of the enzyme on the affinity tagged enzyme substrate, butcarries isotope labels which may include ²H, ¹³C, ¹⁵N, ¹⁷O, ¹⁸O, or ³⁴S,that allow it to be independently detected by MS techniques. Internalstandards for use in method herein to quantitative one or severalproteins in a sample are prepared by reaction of affinity labeledprotein reactive reagents with a known protein to generate the affinitytagged peptides generated from digestion of the tagged protein. Affinitytagged peptides internal standards are substantially chemicallyidentical to the corresponding affinity tagged peptides generated fromdigestion of affinity tagged protein, except that they aredifferentially isotopically labeled to allow their independent detectionby MS techniques.

[0061] The method of this invention can also be applied to determine therelative quantities of one or more proteins in two or more proteinsamples, the proteins in each sample are reacted with affinity taggingreagents which are substantially chemically identical but differentiallyisotopically labeled. The samples are combined and processed as one. Therelative quantity of each tagged peptide which reflects the relativequantity of the protein from which the peptide originates is determinedby the measurement of the respective isotope peaks by mass spectrometry.

[0062] The methods of this invention can be applied to the analysis orcomparison of multiple different samples. Samples that can be analyzedby methods of this invention include cell homogenates; cell fractions;biological fluids including urine, blood, and cerebrospinal fluid;tissue homogenates; tears; feces; saliva; lavage fluids such as lung orperitoneal ravages; mixtures of biological molecules including proteins,lipids, carbohydrates and nucleic acids generated by partial or completefractionation of cell or tissue homogenates.

[0063] The methods of this invention employ MS and (MS)^(n) methods.While a variety of MS and (MS)^(n) are available and may be used inthese methods, Matrix Assisted Laser Desorption Ionization MS (MALDI/MS)and Electrospray Ionization MS (ESI/MS) methods are preferred.

[0064] Quantitative Proteome Analysis

[0065] This method is schematically illustrated in Scheme 1 using abiotin labeled sulfhydryl-reactive reagent for quantitative proteinprofile measurements in a sample protein mixture and a reference proteinmixture. The method comprises the following steps:

[0066] Reduction. Disulfide bonds of proteins in the sample andreference mixtures are reduced to free SH groups. The preferred reducingagent is tri-n-butylphosphine which is used under standard conditions.Alternative reducing agents include mercaptoethylamine anddithiothreitol. If required, this reaction can be performed in thepresence of solubilizing agents including high concentrations of ureaand detergents to maintain protein solubility. The reference and sampleprotein mixtures to be compared are processed separately, applyingidentical reaction conditions;

[0067] Derivatization of SH groups with an affinity tag. Free SH groupsare derivatized with the biotinylating reagentbiotinyl-iodoacetylamidyl-4,7,10 trioxatridecanediamine the synthesis ofwhich is described below. The reagent is prepared in differentisotopically labeled forms by substitution of linker atoms with stableisotopes and each sample is derivatized with a different isotopicallylabeled form of the reagent. Derivatization of SH groups is preferablyperformed under slightly basic conditions (pH 8.5) for 90 min at RT. Forthe quantitative, comparative analysis of two samples, one sample each(termed reference sample and sample) are derivatized as illustrated inScheme 1 with the isotopically light and the isotopically heavy form ofthe reagent, respectively. For the comparative analysis of severalsamples one sample is designated a reference to which the other samplesare related to. Typically, the reference sample is labeled with theisotopically heavy reagent and the experimental samples are labeled withthe isotopically light form of the reagent, although this choice ofreagents is arbitrary. These reactions are also compatible with thepresence of high concentrations of solubilizing agents;

[0068] Combination of labeled samples. After completion of the affinitytagging reaction defined aliquots of the samples labeled with theisotopically different reagents (e.g., heavy and light reagents) arecombined and all the subsequent steps are performed on the pooledsamples. Combination of the differentially labeled samples at this earlystage of the procedure eliminates variability due to subsequentreactions and manipulations. Preferably equal amounts of each sample arecombined;

[0069] Removal of excess affinity tagged reagent. Excess reagent isadsorbed, for example, by adding an excess of SH-containing beads to thereaction mixture after protein SH groups are completely derivatized.Beads are added to the solution to achieve about a 5-fold molar excessof SH groups over the reagent added and incubated for 30 min at RT.After the reaction the beads are be removed by centrifugation;

[0070] Protein digestion. The proteins in the sample mixture aredigested, typically with trypsin. Alternative proteases are alsocompatible with the procedure as in fact are chemical fragmentationprocedures. In cases in which the preceding steps were performed in thepresence of high concentrations of denaturing solubilizing agents thesample mixture are diluted until the denaturant concentration iscompatible with the activity of the proteases used. This step may beomitting in the analysis of small proteins;

[0071] Affinity isolation of the affinity tagged peptides by interactionwith a capture reagent. The biotinylated peptides are isolated onavidin-agarose. After digestion the pH of the peptide samples is loweredto 6.5 and the biotinylated peptides are immobilized on beads coatedwith monomeric avidin (Pierce). The beads are extensively washed. Thelast washing solvent includes 10% methanol to remove residual SDS.Biotinylated peptides are eluted from avidin-agarose, for example, with0.3% formic acid at pH 2;

[0072] Analysis of the isolated, derivatized peptides by μLC-MS^(n) orCE-MS^(n) with data dependent fragmentation. Methods and instrumentcontrol protocols well-known in the art and described, for example, inDucret et al., 1998; Figeys and Aebersold, 1998; Figeys et al., 1996; orHaynes et al., 1998 are used.

[0073] In this last step, both the quantity and sequence identity of theproteins from which the tagged peptides originated can be determined byautomated multistage MS. This is achieved by the operation of the massspectrometer in a dual mode in which it alternates in successive scansbetween measuring the relative quantities of peptides eluting from thecapillary column and recording the sequence information of selectedpeptides. Peptides are quantified by measuring in the MS mode therelative signal intensities for pairs of peptide ions of identicalsequence that are tagged with the isotopically light or heavy forms ofthe reagent, respectively, and which therefore differ in mass by themass differential encoded within the affinity tagged reagent. Peptidesequence information is automatically generated by selecting peptideions of a particular mass-to-charge (m/z) ratio for collision-induceddissociation (CID) in the mass spectrometer operating in the MS^(n)mode. (Link, A. J. et al., 1997; Gygi, S. P., et al. 1999; and Gygi, S.P. et al., 1999). The resulting CID spectra are then automaticallycorrelated with sequence databases to identify the protein from whichthe sequenced peptide originated. Combination of the results generatedby MS and MS^(n) analyses of affinity tagged and differentially labeledpeptide samples therefore determines the relative quantities as well asthe sequence identities of the components of protein mixtures in asingle, automated operation.

[0074] Results of this applying this method using the biotinylatedsulfhydryl reagent and to the quantitative analysis of synthetic peptidesamples, to the relative quantitation of the peptides in a proteindigest an the tandem mass spectral analysis of a derivatized peptide areshown in FIG. 1, Table 1, and FIG. 2, respectively.

[0075] This method can also be practiced using other affinity tags andother protein reactive groups, including amino reactive groups, carboxylreactive groups, or groups that react with homoserine lactones.

[0076] The approach employed herein for quantitative proteome analysisis based on two principles. First, a short sequence of contiguous aminoacids from a protein (5-25 residues) contains sufficient information touniquely identify that protein. Protein identification by MS^(n) isaccomplished by correlating the sequence information contained in theCID mass spectrum with sequence databases, using sophisticated computersearching algorithms (Eng, J. et al., 1994; Mann, M. et al., 1994; Qin,J. et al., 1997; Clauser, K. R. et al., 1995). Second, pairs ofidentical peptides tagged with the light and heavy affinity taggedreagents, respectively, (or in analysis of more than two samples, setsof identical tagged peptides in which each set member is differentiallyisotopically labeled) are chemically identical and therefore serve asmutual internal standards for accurate quantitation. The MS measurementreadily differentiates between peptides originating from differentsamples, representing for example different cell states, because of thedifference between isotopically distinct reagents attached to thepeptides. The ratios between the intensities of the differing weightcomponents of these pairs or sets of peaks provide an accurate measureof the relative abundance of the peptides (and hence the proteins) inthe original cell pools because the MS intensity response to a givenpeptide is independent of the isotopic composition of the reagents (DeLeenheer, A. P. et al (1992). The use of isotopically labeled internalstandards is standard practice in quantitative mass spectrometry and hasbeen exploited to great advantage in, for example, the precisequantitation of drugs and metabolites in bodily fluids (De Leenheer, A.P. et al., 1992).

[0077] In another illustration of the method, two mixtures consisting ofthe same six proteins at known, but different, concentrations wereprepared and analyzed. The protein mixtures were labeled, combined andtreated as schematically illustrated in Scheme 1. The isolated, taggedpeptides were quantified and sequenced in a single combined μLC-MS andμLC-MS^(n) experiment on an ESI ion trap mass spectrometer. All sixproteins were unambiguously identified and accurately quantified (Table2). Multiple tagged peptides were encountered for each protein. Thedifferences between the observed and expected quantities for the sixproteins ranged between 2 and 12%.

[0078] The process is further illustrated for a single peptide pair inFIGS. 3A-C. A single scan of the mass spectrometer operated in MS modeis shown in FIG. 3A. Four pairs of peptide ions characterized by themass differential encoded in the affinity tagged reagent are detected inthis scan and indicated with their respective m/z values. The scan shownwas acquired in 1.3 s. Over the course of the one-hour chromatographicelution gradient, more than 1200 such scans were automatically recorded.FIG. 3B shows an expanded view of the mass spectrum around the ion pairwith m/z ratios of 993.8 and 977.7, respectively. Co-elution and adetected mass differential of four units potentially identifies the ionsas a pair of doubly charged affinity tagged peptides of identicalsequence (mass difference of eight and a charge state of two). FIG. 3Cshows the reconstructed ion chromatograms for these two species. Therelative quantities were determined by integrating the contour of therespective peaks. The ratio (light/heavy) was determined as 0.54 (Table1). The peaks in the reconstructed ion chromatograms appear serratedbecause in every second scan the mass spectrometer switched between theMS and the MS^(n) modes to collect sequence information (CID massspectrum) of a selected peptide ion. These CID spectra were used toidentify the protein from which the tagged peptides originated. FIG. 4Ashows the CID spectrum recorded from the peptide ion with m/z=998(marked with an arrow in FIG. 3A). Database searching with this CIDspectrum identified the protein as glyceraldehyde-3-phosphatedehydrogenase (FIG. 4B) which was a member of the protein mixture.

[0079] Several beneficial features of the this method are apparent.First, at least two peptides were detected from each protein in themixture. Therefore, both quantitation and protein identification can beredundant. Second, the identified peptides all contained at least onetagged cysteinyl residue. The presence of the relatively rare cysteinylresidue in a peptide adds an additional powerful constraint for databasesearching (Sechi, S. et al., 1998). Third, tagging and selectiveenrichment of cysteine-containing peptides significantly reduced thecomplexity of the peptide mixture generated by the concurrent digestionof six proteins. For this protein mixture, the complexity was reducedfrom 293 potential tryptic peptides to 44 tryptic peptides containing atleast one cysteinyl residue. Fourth, the peptide samples eluted from theavidin affinity column are directly compatible with analysis byμLC-MS^(n).

[0080] Quantitative Analysis of Protein Expression in Different CellStates

[0081] The protein reactive affinity reagent strategy was applied tostudy differences in steady-state protein expression in the yeast, S.cerevisiae, in two non-glucose repressed states (Table 3). Cells wereharvested from yeast growing in log-phase utilizing either 2% galactoseor 2% ethanol as the carbon source. One-hundred μg of soluble yeastprotein from each cell state were labeled independently with theisotopically different affinity tagged reagents. The labeled sampleswere combined and subjected to the strategy described in FIG. 1. Onefiftieth (the equivalent of approximately 2 μg of protein from each cellstate) of the sample was analyzed.

[0082] Glucose repression causes large numbers of proteins withmetabolic functions significant to growth on other carbon sources to beminimally expressed (Ronne, H., 1995; Hodges, P. E. et al., 1999).Growth on galactose or ethanol with no glucose present results in theexpression of glucose repressed genes. Table 3 presents a selection of34 yeast genes encountered in the analysis, but it contains every knownglucose-repressed genes that was identified (Mann, M. et al., 1994).Each of these genes would have been minimally expressed in yeast grownon glucose. Genes specific to both growth on galactose (GAL1, GAL10) aswell as growth on ethanol (ADH2, ACH1) were detected and quantitated.

[0083] The quantitative nature of the method is apparent in the abilityto accurately measure small changes in relative protein levels. Evidenceof the accuracy of the measurements can be seen by the excellentagreement found by examining ratios for proteins for which multiplepeptides were quantified. For example, the five peptides found from PCK1had a mean ratio ±95% confidence intervals of 1.57±0.15, and the percenterror was <10%. In addition, the observed changes fit the expectedchanges from the literature (Ronne, H., 1995; Hodges, P. E. et al.,1999). Finally, the observed changes are in agreement with the changesin staining intensity for these same proteins examined aftertwo-dimensional gel electrophoresis (data not shown).

[0084] The alcohol dehydrogenase family of isozymes in yeast facilitatesgrowth on either hexose sugars (ADH1) and ethanol (ADH2). The gene ADH2encodes an enzyme that is both glucose- and galactose-repressed andpermits a yeast cell to grow entirely on ethanol by converting it intoacetaldehyde which enters the TCA cycle (FIG. 5A). In the presence ofsugar, ADH1 performs the reverse reaction converting acetaldehyde intoethanol. The regulation of these isozymes is key to carbon utilizationin yeast (Ronne, H., 1995). The ability to accurately measuredifferences in gene expression across families of isozymes is sometimesdifficult using cDNA array techniques because of cross hybridization(DeRisi, J. L. et al., 1997). The method of this invention applied asillustrated in FIG. 1 succeeded in measuring gene expression for eachisozyme even though ADH1 and ADH2 share 93% amino acid (88% nucleotide)sequence similarity. This was because the affinity tagged peptides fromeach isozyme differed by a single amino acid residue (valine tothreonine) which shifted the retention time by more than 2 min and themass by 2 daltons for the ADH2 peptides (FIG. 5B). ADH1 was expressed atapproximately 2-fold high levels when galactose was the carbon sourcecompared with ethanol. Ethanol-induction of ADH2 expression resulted inmore than 200-fold increases compared with galactose-induction.

[0085] The results described above illustrate that the method of thisinvention provides quantitative analysis of protein mixtures and theidentification of the protein components therein in a single, automatedoperation.

[0086] The method as applied using a sulfhydryl reactive reagentsignificantly reduces the complexity of the peptide mixtures becauseaffinity tagged cysteine-containing peptides are selectively isolated.For example, a theoretical tryptic digest of the entire yeast proteome(6113 proteins) produces 344,855 peptides, but only 30,619 of thesepeptides contain a cysteinyl residue. Thus, the complexity of themixture is reduced, while protein quantitation and identification arestill achieved. The chemical reaction in of the sulfhydryl reagent withprotein can be performed in the presence of urea, sodium dodecyl sulfate(SDS), salts and other chemicals that do not contain a reactive thiolgroup. Therefore, proteins can be kept in solution with powerfulstabilizing agents until they are enzymatically digested. Thesensitivity of the μLC-MS^(n) system is dependent of the sample quality.In particular, commonly used protein solubilizing agents are poorlycompatible or incompatible with MS. Affinity purification of the taggedpeptides completely eliminates contaminants incompatible with MS. Thequantitation and identification of low abundance proteins byconventional methods requires large amounts (milligrams) of startingprotein lysate and involves some type of enrichment for these lowabundance proteins. Assays described above, start with about 100 μg ofprotein and used no fractionation techniques. Of this, approximately{fraction (1/50)} of the protein was analyzed in a single μLC-MS^(n)experiment. This system has a limit of detection of 10-20 fmol perpeptide (Gygi, S. P. et al., 1999). For this reason, in the assaysdescribed which employ μLC-MS^(n) only abundant proteins are detected.However, the methods of this invention are compatible with anybiochemical, immunological or cell biological fractionation methods thatreduce the mixture complexity and enrich for proteins of low abundancewhile quantitation is maintained. This method can be redundant in bothquantitation and identification if multiple cysteines are detected.There is a dynamic range associated with the ability of the method toquantitate differences in expression levels of affinity tagged peptideswhich is dependent on both the intensity of the peaks corresponding thepeptide pair (or set) and the overall mixture complexity. In addition,this dynamic range will be different for each type of mass spectrometerused. The ion trap was employed in assays described herein because ofits ability to collect impressive amounts of sequencing information(thousands of proteins can potentially be identified) in adata-dependent fashion even though it offers a more limited dynamicquantitation range. The dynamic range of the ion trap (based onsignal-to-noise ratios) varied depending on the signal intensity of thepeptide pair and complexity of the mixture, but differences of up to100-fold were generally detectable and even larger differences could bedetermined for more abundant peptides. In addition, protein expressionlevel changes of more than 100-200-fold still identify those proteins asmajor potential contributors to the phenotypic differences between thetwo original cell states. The method can be extended to includereactivity toward other functional groups. A small percentage ofproteins (8% for S. cerevisiae) contain no cysteinyl residues and aretherefore missed by analysis using reagents with sulfhydryl groupspecificity (i.e., thiol group specificity). Affinity tagged reagentswith specificities toward functional groups other than sulfhydryl groupswill also make cysteine-free proteins susceptible to analysis.

[0087] The methods of this invention can be applied to analysis of lowabundance proteins and classes of proteins with particularphysico-chemical properties including poor solubility, large or smallsize and extreme p/values.

[0088] The prototypical application of the chemistry and method is theestablishment of quantitative profiles of complex protein samples andultimately total lysates of cells and tissues following the preferredmethod described above. In addition the reagents and methods of thisinvention have applications which go beyond the determination of proteinexpression profiles. Such applications include the following:

[0089] Application of amino-reactive or sulfhydryl-reactive,differentially isotopically labeled affinity tagged reagents for thequantitative analysis of proteins in immuno precipitated complexes. Inthe preferred version of this technique protein complexes from cellsrepresenting different states (e.g., different states of activation,different disease states, different states of differentiation) areprecipitated with a specific reagent, preferably an antibody. Theproteins in the precipitated complex are then derivatized and analyzedas above.

[0090] Application of amino-reactive, differentially isotopicallylabeled affinity tagged reagents to determine the sites of inducedprotein phosphorylation. In a preferred version of this method purifiedproteins (e.g., immunoprecipitated from cells under differentstimulatory conditions) are fragmented and derivatized as describedabove. Phosphopeptides are identified in the resulting peptide mixtureby fragmentation in the ion source of the ESI-MS instrument and theirrelative abundances are determined by comparing the ion signalintensities of the experimental sample with the intensity of anincluded, isotopically labeled standard.

[0091] Amino-reactive, differentially isotopically labeled affinitytagged reagents are used to identify the N-terminal ion series in MS^(n)spectra. In a preferred version of this application, the peptides to beanalyzed are derivatized with a 50:50 mixture of an isotopically lightand heavy reagent which is specific for amino groups. Fragmentation ofthe peptides by CID therefore produce two N-terminal ion series whichdiffer in mass precisely by the mass differential of the reagent speciesused. This application dramatically reduces the difficulty indetermining the amino acid sequence of the derivatized peptide.

[0092] Quantitative Analysis of Surface Proteins in Cells and Tissue

[0093] The cell exterior membrane and its associated proteins (cellsurface proteins) participate in sensing external signals and respondingto environmental cues. Changes in the abundance of cell surface proteinscan reflect a specific cellular state or the ability of a cell torespond to its changing environment. Thus, the comprehensive,quantitative characterization of the protein components of the cellsurface can identify marker proteins or constellations of markerproteins characteristic for a particular cellular state, or explain themolecular basis for cellular responses to external stimuli. Indeed,changes in expression of a number of cell surface receptors such asHer2/neu, erbB, IGFI receptor, and EGF receptor have been implicated incarcinogenesis and a current immunological therapeutic approach forbreast cancer is based on the infusion of an antibody (Herceptin,Genentech, Palo Alto, Calif.) that specifically recognizes Her2/neureceptor.

[0094] Cell surface proteins are also experimentally accessible.Diagnostic assays for cell classification and preparative isolation ofspecific cells by methods such as cell sorting or panning are based oncell surface proteins. Thus, differential analysis of cell surfaceproteins between normal and diseased (e.g., cancer) cells can identifyimportant diagnostic or therapeutic targets. While the importance ofcell surface proteins for diagnosis and therapy of cancer has beenrecognized, membrane proteins have been difficult to analyze. Due totheir generally poor solubility they tend to be under-represented instandard 2D gel electrophoresis patterns and attempts to adapt 2Delectrophoresis conditions to the separation of membrane proteins havemet limited success. The method of this invention can overcome thelimitations inherent in the traditional techniques.

[0095] The analysis of membrane proteins is challenging because theygenerally are difficult to maintain in solution under conditions thatare compatible with high sensitivity analytical instruments such as massspectrometers. The application of the methods of the present inventionto the analysis of membrane proteins is exemplified using human T celllymphoma cell line Jurkat for membrane protein labeling and extractionand the well characterized human prostate epithelial cell line P69SV40Tand two P69SV40T sublines which differ in IGF-1 receptor expression byfactor of 10 to exemplify quantitative, differential analysis ofmembrane proteins.

[0096] Jurkat cells are an appropriate model system because the cellsare easy to grow in large numbers and because the modulation of cellsurface proteins in response to different stimuli and experimentalconditions has been well characterized in T lymphocytes. Commerciallyavailable biotinylating reagents or more generally affinity taggingreagents are employed to derivatize lysine residues and the freeN-termini. Water soluble biotinylating reagents such as Sulfo-NHS(N-hydroxy succinimide) biotin and analogs(Sulfosuccinimidyl-6-(biotinamido)-hexanoate, Pierce, Rockford, Ill.)which have been used extensively for labeling cell surface proteins canbe employed. The reaction of NHS esters with primary amines is best atneutral pH values and above and is compatible with the presence oforganic solvent such as DMSO or DMF. Biotinylation of cell surfaceproteins from the Jurkat cells is carried out in PBS buffer at pH 7.2.Cells (1×10⁷) are washed with PBS buffer to remove contaminating serumand other proteins from the culture medium. The cells are resuspended at25×10⁶ cell/ml and reacted with 0.5 mg/ml of Sulfo-NHS-Biotin (Pierce,Rockford, Ill.) for 30 min at RT. The labeled cells are washed twicewith cold PBS to remove unreacted biotinylating reagent. Biotinylatedcells are solubilized at 5×10⁷ cells/ml in lysis buffer containing 1%Triton X-114. Triton X-114 has the property of phase-partitioning intodetergent phase and aqueous phase at 30° C. Following the phasepartitioning, detergent phase is removed from the aqueous phase bycentrifugation at 300× g. Phase partitioning has previously beensuccessfully used to enrich cell membrane. Also, this technique wasfound to enrich membrane proteins from Jurkat cell lysates. Triton phaseis diluted 1:5 (v/v) using 50 mM ammonium bicarbonate buffer, pH 8.5,and high-purity, modified porcine-trypsin is added to digest theproteins at a concentration of 12.5 ng/ml for overnight at 37° C.Trypsin is neutralized by the addition of a cocktail of serine proteaseinhibitors and tryptic peptides are isolated by the avidin affinitychromatography techniques. Eluted peptides are separated e.g., by μLCmethods and identified by searching peptide sequence databases, usingfor example, the Sequest program.

[0097] The human prostate epithelial cell line P69SV40T which wasimmortalized with SV 40 T antigen has been well characterized. This cellline is immortal but not tumorigenic and expresses type 1 insulin likegrowth factor receptor (IGF-1R) at 2×10⁴ receptors per cell. A subline,called M12, was derived from P69SV40T by sequential passage in maleathymic nude mice. This cell line is highly tumorigenic and metastaticand expresses 1.1×10³ IGF-1R per cell. The relative difference in theabundance of IGF-1R in the cell lines P69SV40T and M12 can bequantitatively determined using methods of this invention adapted forapplication to membrane proteins. Since the number of IGF-1R for thesecell lines has already been determined, this well characterized systemcan provide a reference to validate the efficiency of the quantitativemethods of this invention

[0098] P69SV40T cells (1×10⁷) are biotinylated with an isotopicallyheavy biotin tagged amino reactive reagent and the M12 cells (1×10⁷) arebiotinylated with a corresponding isotopically light amine reactivebiotin tagged amino reactive reagent. IGF-1R is then immunoprecipitatedfrom the combined lysate of both cell lines using an antibody againsthuman IGF-1R and the total mass of immunoprecipitated proteins isdigested with trypsin. Trypsin is then neutralized, e.g., by theaddition of inhibitors and tagged peptides are purified by biotin-avidinaffinity chromatography. The eluted peptides are analyzed by LC-MS andLC-MSN for peptide quantitation and identification, respectively, as hasbeen described above. Quantitation in this experiment is facilitated bythe option to use selective ion monitoring in the MS. In this mode onlythe masses of tagged peptide ions expected to derive from IGF-1R need bemonitored.

[0099] The described technique can be applied to compare the differencesin relative abundance of cell surface proteins between parental prostatecell line (P69SV40T) and M12 cells to detect and identify those cellsurface proteins whose expression level is different in the two celllines and which may be characteristic of the different cell states.Using the methods described herein global, relative quantitation of thecell surface proteins in any two or more cell lines can be analyzed todetect and identify those cell surface proteins characteristic of thedifferent cell states. Results can be independent confirmed usingprocedure such as 1D or 2D gels, if applicable, or quantitative westernblotting to confirm quantitation results.

[0100] It is expected that the experimental variability of quantitationof cell surface proteins will be considerably better than the accuracyof quantitation achieved by currently available cDNA array technology.In addition to relative protein quantity and identity, the method canalso be used to reveal the orientation of the protein in the membrane,based on the presumption that intact, alive cells will exclude thebiotinylating reagent.

[0101] Alternative methods can be applied to enhance the selectivity fortagged peptides derived from cell surface proteins. For example, taggedcell surface proteins can be trypsinized directly on the intact cells togenerate tagged peptides, purified and analyzed as discussed. Inaddition, traditional cell membrane preparations may be used as aninitial step to enrich cell surface proteins. These methods can includegentle cell lysis with a dounce homogenizer and series of densitygradient centrifugations to isolate membrane proteins prior toproteolysis. This method can provide highly enriched preparations ofcell surface proteins. Affinity tagged proteins may also be isolated byaffinity chromatography prior to proteolysis as well as afterproteolysis. This chromatography can be performed in the presence ofsurfactants such as TX-100, NP-40 or Tween-20 to maintain proteinsolubility. The sequential application of affinity chromatography steps(one for the intact protein and one for the tagged peptide fragments)provides a high degree of selectivity. These alternative methods areeasily scalable for the detection of low abundance membrane proteins andthe relative quantity of tagged peptides tagged is maintained throughthe selective enrichment steps.

[0102] In the application of the methods of this invention to cellsurface proteins, once the tagged proteins are fragmented, the taggedpeptides behave no differently from the peptides generated from moresoluble samples.

[0103] Synthesis of Affinity Tagged Protein Reactive Reagents that areSelective for Certain Proteins Groups

[0104] Synthetic routes exemplary affinity tagged reagents suitable foruse in the methods of this invention are provided in Schemes 2-3 wherewell-known synthetic techniques are employed in synthesis of thenon-deuterated and deuterated reagents.

[0105] Biotinyl-iodoacetylamidyl-4,7,10 trioxatridecanediamine 4 (Scheme3) consists of a biotin group, a chemically inert spacer of capable ofbeing isotopically labeled with stable isotopes and a iodoacetamidylgroup, respectively. The biotin group is used for affinity enrichment ofpeptides derivatized with the reagent, the ethylene glycol linker isdifferentially isotopically labeled for mass spectral analysis and theiodoacetamidyl group provides specificity of the reagent forsulfhydryl-containing peptides. The reagent an be synthesized in an allhydrogen form (isotopically light form) with and with 1-20, andpreferably 4-8 deuterium atoms in the linker (isotopically heavy forms).

[0106] Analysis of Velocities of Multiple Enzymes in Cell Lysates

[0107] Monitoring enzyme functions by biochemical assays is an essentialdiagnostic tool that employs a multitude of analytical techniquesincluding spectrophotometric, fluorometric, and radiometric detection ofproducts. However, current methods are difficult to use for assayingseveral enzymes simultaneously in a single sample. Mass spectrometry forquantification of a collection of metabolites in biological fluids hasemerged as a powerful approach for the analysis of birth defects (Morriset al., 1994), but this analytical technique has not been developed forthe direct analysis of rates of individual enzymatic steps. Theanalytical method described herein for monitoring and quantification ofenzymatic activities in cell homogenates and other biological samplespermits simultaneous (multiplex) monitoring of multiple reactions, andcan be readily automated.

[0108] A feature of the method of this invention as applied to enzymeassays is the use of electrospray ionization mass spectrometry (ESI-MS)(Cole et al., 1997) for the simultaneous detection of enzymatic productsand chemically identical internal standards, which are distinguished bystable isotope (deuterium) labeling. A second feature is the use ofaffinity tagged reagents containing an enzyme substrate which whencombined with affinity purification provide for facile capture ofenzymatic products from crude biological fluids. The affinity taggedreagents are designed to contain a target substrate for an enzyme ofinterest that is covalently attached to an affinity tag via a linker.Action of the enzyme of interest on the substrate conjugate causescleavage or other modification that changes its molecular mass (Scheme4). The change of mass is detected by ESI-MS. The linker and affinitytag used preferably facilitate ionization by ESI, block action of otherenzymes in the biological fluid, and allow highly selective capture fromthe complex matrix for facile purification.

[0109] An example of this approach is the design and synthesis ofaffinity tagged enzyme substrate reagents 1 and 2 (Scheme 5) tosimultaneously assay lysosomal β-galactosidase andN-acetyl-α-D-glucosaminidase, respectively. Deficiency of the formerenzyme results in one of the lysosomal storage diseases,GM₁-gangliosidosis, a condition that occurs in the population with afrequency of about 1 in 50,000 and leads to early death of affectedchildren. Deficiency of N-acetyl-R-D-glucosaminidase results in the rarelysosomal storage disorder Sanfilippo syndrome type B. This example hasbeen described in Gerber et al., 1999, which is incorporated byreference herein in its entirety.

[0110] Conjugates 1 and 2 consist of biotin as an affinity tag, which iscoupled to sarcosine. Biotin allows highly specific capture of thesubstrate conjugate through non-covalent binding to streptavidinimmobilized on agarose beads (Bayer et al., 1990). Sarcosine provides anN-methylated amide linkage to biotin to block the enzyme biotinidase,which is often present in the cellular fluids and could cause cleavageof the conjugate molecule during the assay (Wilbur et al., 1997). Inaddition, it was found that biotinyl-sarcosine conjugates can bedisplaced from streptavidin by addition of biotin. TheN-biotinylsarcosine block is linked to a polyether diamine, the lengthof which can be varied to avoid mass/charge overlaps of products andinternal standards. The linker also allows facile introduction ofmultiple deuterium atoms (i.e., 8 deuteriums in 5 and 4 in 6, Scheme 5)to permit the synthesis of internal standards. The d8-linker was made byreacting DOCH₂CH₂OCH₂CH₂OD with CD₂═CDCN in benzene with catalytic NaOD(Ashikaga, K., et al., 1988) and the resulting dinitrile was reduced tothe diamine with Ra—Ni. The d4-linker was made in the same way usingethylene glycol and CD2═dCDCN in CH₃CN and catalytic NaOH.

[0111] In addition, the linker is hydrophilic to ensure good watersolubility of the substrate conjugate, and it has basic groups which areefficiently protonated by ESI and thus ensure sensitive detection bymass spectrometry. The target carbohydrate substrates are attached tothe polyether linker by a β-alanine unit (Scheme 5). The enzymaticproduct conjugates 3 and 4 are also shown Scheme 5. Conjugates 1 and 2were prepared as shown in Scheme 5. All reagents were purified tohomogeneity by reverse-phase HPLC and characterized by high-field 1H NMRand ESI-MS. The substrate was linked to the diamine spacer by Michaeladdition of the latter onto the p-acryloylamidophenyl glycoside,(Romanowska et al., 1994) and the intermediate was coupled with thetetrafluorophenyl ester of N-biotinylsarcosine (Wilbur et al., 1997).

[0112] The ESI-MS assay of βgalactosidase andN-acetyl-R-D-glucosaminidase is based on enzymatic cleavage of theglycosidic bond to release monosaccharide and conjugates 3 and 4 (massdifferences are 162 and 203 Da, respectively). In a typical procedure,0.2 mM 1 and 0.3 mM 2 were incubated with sonicated cultured fibroblastsfrom individual patients with β-galactosidase deficiency and withfibroblasts cultured from unaffected people. After incubation, labeledinternal standards 5 and 6 were added, and the biotinylated componentswere captured on streptavidin-agarose beads. Quantitative strepavidincapture efficiency from a cell homogenate was observed with modelreagents. After purification by multiple washings to removenonspecifically bound components, the biotinylated products werereleased by free biotin, and the eluant was analyzed by ESI-MS. About85% release of the biotinylated products was observed after incubationwith excess biotin for 90 min. A blank was obtained by quenching theassay with all components present at time zero.

[0113] A typical procedure, cell protein (75 μg) in 15 μL of water wasadded to 15 μL buffer (0.1 M Na citrate, pH 4.25) containing 2 (0.3 mM)and 1 (0.2 mM, added 5 h after addition of cell protein). Afterincubation for 5.5 h at 37° C., the reaction was quenched by addition of200 μL of 0.2 M glycine carbonate buffer, pH 10.3, and 5 and 6 (1 nmoleach) were added. After centrifugation to remove cell debris, thesupernatant was loaded onto a bed of streptavidin-agarose (7 nmol biotinbinding capacity, Pierce) in a small filtration device (micro BioSpin,Bio-Rad). After 5 min, filtration was effected by centrifugation, andthe gel bed was washed with 0.1% Triton X-100 (about 1 min incubation,then spin) and then six times with purified water (Milli-Q, Millipore).Elution was carried out in 25 μL of 50% methanol containing 56 nmol offree biotin (1-10 h incubation, then spin). Filtrate was diluted 4-foldwith 50% methanol/water, and 1 μL was analyzed by ESI-MS.

[0114] The ESI-MS spectrum of the blank (FIG. 6A) is remarkably simple,showing peaks of the (M+H)⁺ ions from reagents 1 and 2 (m/z 843 and840), internal standards 5 and 6 (m/z 689 and 641), and trace amounts ofproducts 3 and 4 (m/z 681 and 637). Ions due to clusters of biotin alsoappear in the spectrum but did not interfere with the analysis. Thepresence of nondeuterated products in the blank may be due tononenzymatic substrate reagent hydrolysis during sample work up or tocollision-induced dissociation of the substrate ion in the gas phase. AMS/MS spectrum of the (conjugate 1+H)⁺ ion at m/z 843 gave a prominentfragment of (conjugate 3+H)⁺ at m/z 681 (spectrum not shown). The ESI-MSspectrum of a sample incubated with cell homogenate from a healthyindividual clearly shows the β-galactosidase product at m/z 681 and theN-acetyl-α-D-glucosaminidase product at m/z 637 (FIG. 6B). Triplicateenzymatic reactions using cells from a healthy patient yielded aβ-galactosidase specific activity of 51±3 nmol/h/(mg cell protein) andan N-acetyl-α-D-glucosaminidase specific activity of 1.4±0.3 nmol/h/mg.Time course studies confirmed that the initial reaction velocities werebeing measured. Values obtained with cells from six healthy individualsranged from 36±4 to 68±3 nmol/h/mg for γ-galactosidase and 0.9±0.05 to2.3±0.4 nmol/h/mg for N-acetyl-α-D-glucosaminidase. In contrast, verylittle enzymatic product above the blank level (0.9±0.9 and 0.8±0.6nmol/h/mg) was observed when cells from two patients withγ-galactosidase deficiency were used, whereasN-acetyl-α-D-glucosaminidase activity is clearly detected (FIG. 6C).These spectra were obtained with 0.75 μg of cell protein, correspondingto ˜1000 fibroblasts. Thus the ESI-MS method has very high sensitivityfor biomedical applications.

[0115] ESI-MS was carried out on a Finnigan LCQ ion trap instrument.Data were collected in full scan mode from m/z 625 to 875 by directinfusion at 1.5 μL/min. Specific activities were obtained from the ratioof product to internal standard ion peak areas (averaged over 30 scans).

[0116] The approach described for assaying enzymes using substratereagents and ESI-MS can be broadly applied. The multiplex technique canbe expanded to assay dozens or more enzymes simultaneously in a singlereaction, obviating the need for multiple assays to assist in confirmingdiagnoses of rare disorders. The method can be used to measure severalenzymes simultaneously when evaluating the rate of chemical flux througha specific biochemical pathway or for monitoring biochemical signalingpathways. The affinity tag-capture reagent method for isolation ofaffinity tagged reaction products and substrates from complex mixturesis technically simple and can be readily automated, particular whenbiotin-strepavidin is employed. Because of the high sensitivity of theESI-MS detection employed, which requires only sub-microgram quantitiesof the substrate reagents per assay, the synthesis of several hundredsubstrate reagents on a low-gram scale becomes practical and economical.Since most enzyme active sites are exposed to solvent, it is possible toattach an affinity tagged linker to most enzyme substrates whilepreserving enzymatic activity. Scheme 6 provides the structures ofseveral additional enzyme substrates, suitable for use in this method,indicating by arrows allowable positions for tag attachment sites.Allowable tag sites for additional enzyme substrates can be determinedby review of X-ray crystal structures of enzyme-substrate orenzyme-substrate analog structures. Using a standard computer graphicsprogram, available X-ray data and by attaching an extended chain butylgroup (as a model for the affinity tagged linker) to potential tagattachment sites, suitable attachment sites that show there are noenzyme-atoms in van der Waals overlap with the model tag can bepredicted.

[0117] Analogous methods to those described above can be applied to theanalysis of enzymes associated with other Sanfillipo Syndromes (A, C andD). SFA is associated with heparin sulfamidase, SFC is associated withacetyl-CoA-alpha-glucosaminide N-acetyltransferase and SFD is associatedwith N-acetylglucosamine 6-sulfatase. Exemplary affinity tagged enzymesubstrate reagents useful in the analysis of these enzymes and thediagnosis of these disorders are provided below. The methods can also beapplied of the diagnosis of Niemann-Pick Type A and B disease byassaying for acid sphingomyelinase and to the diagnosis of Krabbedisease by assaying for galactocerebroside beta-galacatosidase. Theseenzymes are currently assayed employing fluorophore-derivatized reagentsas indicated in Scheme 7. Enzyme substrate reagents for assay of theseenzymes in the methods herein can be readily prepared by replacement ofthe fluorophore with an A—L group herein. This approach to preparationof affinity tagged enzyme substrates is generally applicable to anyknown fluorophore-derivatized enzyme substrate or substrate analog.

[0118] Table 4 provides exemplary enzymes that are associates withcertain birth defects or disease states. These enzymes can be assayed bythe methods described herein.

[0119] Assaying Enzymatic Pathways for Carbohydrate-DeficientGlycoprotein Syndromes (CDGS)

[0120] The methods and reagents of this invention can be employed toquantify the velocities of multiple enzymes pertinent to diagnosis ofCDGS diseases.

[0121] CDGS Type Ia and Ib are caused by the deficiency or absence ofthe enzymes phosphomannoisomerase (PMIb) (Type Ib) andphosphomannomutase (PMM2) (Type Ia) which are part of a multisteppathway (Scheme 8) for conversion of glucose to mannose-1-phosphate(Freeze, 1998). The monosaccharide substrates involved in the pathwayare fructose-6-phosphate, mannose-6-phosphate, and mannose-1-phosphate.These monosaccharides can be somewhat difficult to convert to substrateconjugates because it is not a priori clear which atom on the sugarshould be conjugated with the linker without impairing enzyme activity.PMIb and PMM2 can, however, be assayed indirectly. Mammalian cellmicrosomes contain dolichol-P-mannose synthase which catalyzes thereaction of dolichol-phosphate with GDP-mannose to formdolichol-P-mannose and GDP (Scheme 8, Chapman et al. 1980). Thissynthase can be assayed using the methods of this invention,specifically with a biotin-linker substrate. Microbial PMM and theenzyme which makes GDP-mannose from GTP and mannose-1-P, GDP-mannosepyrophosphorylase, are readily purified from bacteria and yeast (Glaser,1966, Preiss, 1966), and these enzymes can be supplied exogenously tothe enzyme assay. If PMIb activity is deficient, little or nomannose-6-P will be made when the reaction sequence is started byaddition of fructose-6-P. Without mannose-6-P, mannose-1-P andGDP-mannose will not be made, and thus no conjugated-dolichol-P-mannosewill be detected by ESI-MS. Exogenous GTP is supplied as a requirementfor the GDP-mannose pyrophosphorylase step, and ATP is omitted so thatmannose-6-P cannot be made from mannose. To assay PMM2, the reactionsequence is initiated with mannose-6-P, and PMM2 deficiency results inthe failure to make conjugated-dolichol-P-mannose.

[0122] The carrier dolichol is a ˜60- to 105-carbon isoprenoid. Evidenceis accumulating that many enzymes that operate on carbohydrates attachedto dolichol chains are tolerant to significant shortening of thedolichol chain; even 10- and 15-carbon dolichols are tolerated (Rush andWachter, 1995). It appears that such enzymes act on the water-solublecarbohydrate portion of the dolichol conjugate and thus have little orno requirement to bind the dolichol anchor. Based on this, an affinitylabeled substrate for the direct assay of dolichol-P-mannose synthaseand the indirect assay of PMIb and PMM2 is prepared by attaching anaffinity labeled linker to the non-polar end of a short dolichol, suchas the 10-carbon dolichol analog citronellol.

[0123] The synthesis of a biotinylated dolichol₁₀-substrate conjugatecontaining a sarcosinyl linker (B—S—Dol₁O—P) is shown in Scheme 9.Protected citronellol R=t-BuSiMe₂) is regioselectively oxidized at theterminal alyllic methyl group (McMurry and Kocovsky, 1984), and theallylic alcohol is coupled with biotinylsarcosine active ester R═CH₃).The citronellol 1-hydroxy group is subsequently deprotected andphosphorylated with POCl₃ (Rush and Wachter, 1995). In a parallelsynthesis, d₅-sarcosine, CD₃NHCD₂COOH, is used to prepare theisotopically labeled (heavy) reagent for use as an internal standard.d₅-Sarcosine is readily prepares form commercially available materials(BrCD₂COOD and CD₃NH₂) using standard synthetic techniques.

[0124] The deuterated internal standard, B—d₅—S—Dol₁₀—P-Mannose, issynthesized enzymatically by incubating hen oviduct microsomes withGDP-mannose and the synthetic B—d₅—S—Dol₁₀—P substrate conjugate (Rushand Waechler, 1995). An added advantage of the B—S-conjugate is that itallows for a facile affinity purification of the microsomal mannosylatedproduct by specific capture on agarose-streptavidin beads followed byelution with free biotin.

[0125] This method employing affinity tagged short dolichol analogues isgenerally applicable for assaying other enzymes that operated ondolichol anchored carbohydrates. Such an approach is useful for thesubsequent identification of enzyme deficiencies present in other typesof CDGS that have not been yet identified.

[0126] CDGS Type II results from defective GIcNAc transferase II(GIcNAc-T II) which transfers GIcNAc from UDP-GIcNAc to the 2-positionof a mannose residue in the intermediate branched oligosaccharide (theCore Region) in the process of building up the disialo-biantennary chain(Scheme 10) (Schachter, 1986; Brockhausen et al, 1989). GIcNActransferase II is one of the six known enzymes that mediate highlyregiospecific glycosylation of the mannose residues in the Core Region.The Core Region is anchored at the reducing end tochitobiosylasparagine, where the asparagine residue is part of thepeptide chain of the glycosylated protein. The latter structure unit inthe substrate can be replaced by a hydrophobic chain without loss ofenzyme activity (Kaur et al., 1991). Thus, the substrate conjugate forCDGS Type II is assembled by linking a affinity-labeled linker group tothe reducing end to chitobiosylasparagine. However, the latter structureunit in the substrate can be replaced by a hydrophobic chain withoutloss of enzyme activity (Kaur et al., 1991). For example, commerciallyavailable α-D-manno-pyranosylphenylisothiocyanate can be coupled to abiotin-labeled linker and the 5,6-hydroxyls are selectively protected asillustrated in Scheme 11 (Paulsen and Meinjohanns, 1992). Coupling ofthe equatorial 3—OH with per-O-acetylmannosyl-1-trichloroacetamidate(Paulsen et al, 1993) will provide a disaccharide conjugate (Scheme 12).If a minor amount of coupling occurs at the axial 2—OH group theproducts can be separated by HPLC. After deprotection, the primary 6—OHis coupled with a second equivalent ofper-O-acetylmannosyl-1-trichloroacetamidate to yield the Core Regionconjugate. Deprotection of the O-acetyl groups yields the substrateconjugate for GIcNAc transferase I which can be converted to theGIcNAc-T II substrate by enzymatic glycosyl transfer using a TritonX-100 rabbit liver extract, a reaction that has been carried out on apreparative scale (Kaur et al., 1981).

[0127] The synthesis of the deuterium labeled derivative needed for theinternal standard is performed in parallel by using a labeledPEG-diamine building block (Gerber et al., 1999). The biotinylatedtrisaccharide is converted to the tetrasaccharide (product of GIcNAc-TII) by incubation with UDP-GIcNAc and transferase II ((Kaur andHindsgaul, 1991; Tan et al., 1996) and utilizing the B—S handle foraffinity purification of the enzymatic products.

[0128] CDGS Type V

[0129] The lipid-linked oligosaccharide (LLO) that is transferred to theAsn residue of the glycosylated protein is composed of 2 GIcNAc, 9mannoses, and 3 glucoses. It has recently been shown that microsomesfrom CDGS type V patients are greatly deficient in the enzyme thattransfers one or more glucose residues during LLO biosynthesis (Korneret al, 1998). Since the transferase that attaches the carbohydrate unitof LLO to the Asn residue discriminates against the glucose-deficientLLO, CDGS Type V patients have fewer numbers of carbohydrate unitsattached to glycoproteins, such as transferrin (Korner et al., 1998).However, the few carbohydrate units that are present are full-length,demonstrating that residual glucosyl transfer occurs in type V CDGSpatients (Korner et al., 1998). Thus, quantification of the rate of Asnglycosylation by ESI-MS would constitute a viable assay of CDGS Type Vsyndrome.

[0130] Synthetic peptides with 3-7 amino acid residues containing theAsn—Xaa—Ser/Thr sequence have been shown to be good substrates forglycosylation (Ronin et al., 1981). The strategy for the ESI-MS assay ofthe oligosaccharide transferase relies on a B—S conjugate of anappropriate peptide containing the Asn—Xaa—Ser/Thr sequence (Scheme 13).A heptapeptide, NH₂—Tyr—Gln—Ser—Asn—Ser—Thr—Met—NH₂ (SEQ ID NO:1) hasshown high activity in a previous study (Ronin et al., 1981). Thepeptide is readily available by standard peptide synthesis using anin-house automatic synthesizer. The heptapeptide and its glycoconjugatescan be ionized by ESI to provide stable singly-charged ions. Coupling ofBS-tetrafluorophenyl ester with NH₂—Tyr—Gln—Ser—Asn—Ser—Thr—Met—NH₂ willdirectly yield the substrate for the transferase. Several products areexpected from the enzymatic glycosylation and subsequent modificationsof the oligosaccharide antenna. The products can be preparedenzymatically by incubating thyroid rough microsomes withBS—Tyr—Gln—Ser—Asn—Ser—Thr—Met—NH₂ and Dol—P—Glu (Ronin et al., 1981a),followed by affinity purification of the biotinylated products. Productdistribution due to different degrees of glycosylation can be monitoredby ESI-MS, and the major components can be purified by HPLC. Ananalogous procedure using a B—N(CD₃)CD₂CO— conjugate is used to preparedeuterated internal standards.

[0131] The molecular masses of the ionized substrate conjugates for theset of enzymes assayed for CDGS Ia, Ib, II, and V syndromes, as well asproducts and internal standards are compiled in Table 5, which showsthat no isobaric overlaps among the (M+H)⁺ species occur. The closespacing between the (M+Na)+ion from the Type Ia, b product and the(M+H)⁺ ion of the demannosylated B—(N—C₂D₅)—2,2—D₂—Gly—Dol₁₀—P internalstandard can be readily avoided by adjusting the ESI-MS conditions byaddition of Na⁺ ions to generate the gas phase ions as Na-adducts.

[0132] All three of the targeted enzymes can be analyzed simultaneouslyin a single biological sample, such as a cell lysate. The PMM2 and PMIbcannot be assayed simultaneously because they require the addition ofdifferent exogenous substrates. Nevertheless, two assays using identicalESI-MS techniques can be used for diagnosing the various CDGS typesinstead of relying on a battery of different methods.

[0133] Clinical Enzymology Assays

[0134] A fibroblast cell pellet is thawed on ice. Sufficient 0.9% NaClis added to give a final protein concentration in the lysate of ˜5 mg/mL(typically 100 mcL), and the cell pellet is sonicated in ice water 5times for 2 seconds each at moderate power. Total protein is determinedspectrophotometrically using the BCA reagent (BCA Protein Assay kit,Pierce).

[0135] The total enzyme reaction volume is 20-30 mcL. The substratestock solutions are maintained at concentrations of 3 mM (SFB) and 2 mM(GM1). These concentrations were measured by 1H-NMR signal integrationversus an internal standard (formamide proton of DMF). Finalconcentration of substrates is 0.3 and 0.2 mM, respectively. A volume ofreaction buffer (e.g. 200 mM sodium citrate, pH 4.5) equal to thedifference of the substrate addition (2-3 mcL) plus sufficient cellsample volume to equal 50 û 75 mcg total protein from 20-30 mcL is addedto a 0.5 mL Eppendorf tube, followed by substrate. The sample is cooledon ice, and patient cell sample is added. The reaction is initiated byincubation at 37° C.

[0136] For SFB: The reaction is allowed to proceed for 4.5-6 hours,after which GM1 substrate can be added or the reaction can be quenchedwith 100 mcL of 200 mM glycine-carbonate buffer, pH 10.5.

[0137] For GM1: The reaction is allowed to proceed for 0.5 hours.Quenching is as for SFB.

[0138] After quenching, the samples are placed on ice. Internalstandards are added (1 nmol each, i.e. 50 mcL of a 0.02 mM solution).The samples are microfuged at ˜15,000 rpm for 2 min at room temperatureto pellet cell debris. Streptavidin-Agarose beads (Immunopureimmobilized streptavidin, Pierce) are placed in a micro bio-spinchromatography column (Bio-Rad). Sufficient beads are added to give atotal biotin binding capacity of 5 nmol (typical binding capacity 100pmol per mcL of beads as determined by Pierce). The sample supernatantis transferred to the bio-spin tube and allowed to bind for 10 minutesat room temperature. The sample is spun at ˜3,000 rpm to remove excesssupernatant, then washed once with 0.01% Triton X-100 and at least fivetimes with purified water, spinning the tube in-between to removesolution. For each wash, sufficient wash solution is added to fill thebio-spin tube.

[0139] The purified beads are then treated with 30 mcL purified water,followed by 10 mcL of a 4 mM biotin solution. The tubes are capped atthe bottom to prevent leakage and allowed to incubate at 2-8° C. for2-12 hours. The samples are spun at 3,000 rpm to elute the sample into aclean Eppendorf tube.

[0140] The sample is then diluted with 60 mcL of 50% methanol/water andinfused into the ion-trap mass spectrometer. The ESI-MS spectrum istuned to reduce non-specific cleavage of the samples by first analyzinga blank sample (cell lysate added after reaction quench). The infusedsample is analyzed by ion chromatogram integration of a 1 amu-widewindow about the (M+H+)⁺ ions of product and internal standard.

[0141] Results are reported in nmol product formed/hour ofincubation/milligrams total protein in reaction mixtures.

[0142] Clinical Analysis of Patient Samples for GM1 and SFB

[0143] Patient skin fibroblasts were obtained as frozen pellets, andstored at û20° C. until use. Two GM1 affected samples and six normalcontrols were analyzed.

[0144] 50 mcL of 0.9% NaCl was added to each patient cell pellet. Thepellets were lysed by sonication in ice water 5× for 2 seconds each atmoderate sonication power, chilling the microtip in ice water in betweensonications.

[0145] Samples were quantitated by BCA (Pierce) assay as follows:

[0146] Reagent A and B were mixed in 50:1 ratio as described. A proteinstandard curve was prepared using bovine serum albumin as a standard atconcentrations of 2, 1, 0.5, 0.2, and 0.05 mg/mL. A portion of thepatient sonicates were diluted 1:15 in water, and 5 mcL of each dilutedpatient sample and standard curve point was added to separate glassculture tubes containing 200 mcL water, in duplicate. The samples werethen diluted with 1 mL of the mixed BCA reagent, vortexed to mix, andincubated at 37° C. for 60 minutes. The samples were allowed to cool toroom temperature, and analyzed against a blank containing only 200 mcLwater. The samples were analyzed by monitoring absorbance at 562 nm inpolystyrene cuvettes. Average patient absorbance values were blankcorrected and compared to standards via linear regression.

[0147] The patient protein concentrations were determined to be:

[0148] 1.(Affected) 12.2 mg/mL, 2. (Normal) 10.8 mg/mL, 3. (Normal) 11.9mg/mL, 4. (Normal) 12.1 mg/mL, 5. (Normal) 10.3 mg/mL, 6. (Normal) 7.79mg/mL, 7. (Normal) 15.7 mg/mL, 8. (Affected) 11.4 mg/mL

[0149] Incubations were performed in a total of 30 mcL of total volume.The amount of reaction buffer (200 mM sodium citrate, pH 4.25) added toblank Eppendorf tubes was the difference of the substrate volume (3 mcLof each substrate stock solution, 2 mM for GM1 and 3 mM for SFB, for atotal of 6 mcL) plus the volume of cell lysate required to equal 75 mcgtotal protein, from 30 mcL. For example, patient 1. incubation mixtureinitially contained 3 mcL of SFB substrate solution, 6.14 mcL patientcell lysate, and 17.86 mcL reaction buffer. The GM1 substrate was addedlater in the incubation (see below).

[0150] Each patient sample was analyzed in triplicate. The reactionmixtures were kept on ice during preparation, and the reaction wasinitiated by transfer to a 37° C. water bath. 5.5 hr into theincubation, 3 mcL GM1 substrate was added to each reaction, and after anadditional 0.5 hours the reactions were placed on ice and quenched with200 mcL of a 200 mM glycine-carbonate buffer, pH 10.25.

[0151] The purification and analysis procedures are as described inClinical Enzymology Assay (Typical).

[0152] The resultant enzyme activities, as an average standard deviationnmol product/hour incubation/mg total protein: B-Gal SFB Normals RATE+/− SD RATE +/− SD Patient 2 68.0 2.6 0.90 0.05 Patient 3 35.5 3.9 1.540.38 Patient 4 51.1 2.7 1.36 0.26 Patient 5 38.8 8.3 1.01 0.12 Patient 651.4 9.9 2.25 0.36 Patient 7 40.9 3.7 1.12 0.20 Affecteds GM₁ (#1) 0.90.9 0.80 0.21 GM₁ (#8) 0.8 0.6 0.70 0.20

[0153] The following synthetic methods refer to Schemes 14-23.

[0154] Synthesis for GM1-Gangliosidosis (Beta-D-GalactosidaseDeficiency)

[0155] 1. 2,3,5,6-Tetrafluorophenyl trifluoroacetate (1) 25 g (0.15 mol)2,3,5,6-tetrafluorophenol, 35 mL (0.2 mol) trifluoroacetic anhydride and0.5 mL boron trifluoride etherate were refluxed for 18 hours under argonatmosphere. Trifluoroacetic anhydride and trifluoroacetic acid wereremoved by distillation at room temperature. The trifluoroaceticanhydride fraction was returned to the mixture, and the reaction wasrefluxed for 24 hours. This was repeated twice. After final distillationat room temperature, the desired product 1 was distilled at reducedpressure (62° C./45 mmHg) to produce a colorless liquid (30 g,82%).1H-NMR. (Gamper, H. B., 1993). Biotin-2,3,5,6-tetrafluorophenylester (2) A 2.5 g (10.3 mmol) quantity of d-biotin in 20 mL anhydrousDMF under argon atmosphere was warmed to 60° C. with stirring to effectdissolution. 1.7 mL (12.5 mmol) triethylamine was added, followed by 3.4g (12.5 mmol) 1. The mixture was stirred for 2 hours, after which thesolvent was removed by rotary evaporation. The resultant semi-solid wastriturated with 15 mL ether twice to produce a white solid (2.6 g, 65%).1H-NMR. (Wilbur, D. S., et al., 1997). N-methylglycylbiotinamide-methylester (3) A 2.5 g (6.4 mmol) quantity of biotin tetrafluorophenyl esterin 30 mL anhydrous DMF under argon atmosphere was added to a mixture of1.1 g (7.7 mmol) N-methylglycine methyl ester hydrochloride dissolved in10 mL anhydrous DMF and 1.25 mL (9.0 mmol) triethylamine. The reactionmixture was stirred at room temperature for 2 hours, then the solventwas removed by rotary evaporation. The residue was extracted withchloroform (2×100 mL), washed with water (2×20 mL), and dried withanhydrous sodium sulfate. The solvent was removed under vacuum to yield2.1 g (98%) of methyl ester of N-methylglycine biotinamide as an oil.1H-NMR. (Wilbur, D. S., et al., 1997).

[0156] 4. N-methylglycylbiotinamide acid (4) N-Methylglycylbiotinamidemethyl ester was hydrolyzed in a mixture of 31 mL MeOH and 10 mL of 1NNaOH at room temperature with stirring for 1 hour. The mixture wasdiluted with 50 mL 50% MeOH/water and neutralized with cation exchangeresin, hydrogen form (AG MP-50, BioRad). The solution was filtered, theresin washed (3×50 mL) with 50% MeOH/water, and the solvents removed byrotary evaporation to yield 1.6 g (90%) of N-methylglycylbiotinamideacid as an off-white solid. 1H-NMR. (Wilbur, D. S., et al., 1997).

[0157] 5. p-Acrylamidophenyl-β-D-galactopyranoside (5) 40 mg (0.15 mmol)p-aminophenyl β-D-galactopyranoside was added to 25 mL methanol and 200mcL triethylamine with stirring. The solution was chilled in an icebath. 53.3 mg (0.6 mmol) acryloyl chloride was dissolved in 5 mL drymethylene chloride and added dropwise to the stirred solution over 5minutes. The reaction was allowed to return to room temperature,followed by 2 hours of stirring. The solution was then treated withsuccessive anion and cation exchange resins (AG MP-1 and AG MP-50,respectively, BioRad) until neutral pH was obtained with moist pH paper.Solvent was removed by rotary evaporation to yield a solid (43 mg, 90%).1H-NMR. (Romanowska, A., et al., 1994). Michael addition product of4,7,10-trioxa-1,13-tridecanediamine and 5 (6) 20 mg (0.07 mmol) 5 wasadded to a stirred solution of 80 mg (0.35 mmol)4,7,10-trioxa-1,13-tridecanediamine in 5 mL 0.2M sodium carbonate, pH10.5 at 37° C. The reaction was allowed to proceed for 3 days, afterwhich the solution was neutralized with dilute trifluoroacetic acid andpurified by reverse-phase HPLC (Vydac C-18 prep-scale column, 6 mL/min.Mobile phase: H₂O (0.08% TFA)/ACN (0.08% TFA)) to give 7.3 mg ofproduct. (Romanowska, A., et al., 1994).

[0158] 7. GM1 substrate conjugate of 4 and 6 (7) A 2.5 mg (7.4 mcmol)quantity of 4 was dissolved in 1.5 mL anhydrous DMF with stirring, underargon atmosphere. 5 mcL triethylamine was added, followed by 2.3 mg (8.8mcmol) 1. The formation of active ester was monitored by silica TLC (5:1CHCl₃/CH₃OH, Rf 0.5, UV) by briefly drying the spotted TLC plate with astream of air. After 25 minutes, the mixture was added to 3.2 mg (5.9mcmol) 6 in 1 mL anhydrous DMF. After 2 hours, the solvent was removedby vacuum centrifugation and the final product was purified byreverse-phase HPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobilephase: H2O (0.08% TFA)/ACN (0.08% TFA)). Yield 4.6 mg. (For analogouschemistry, see Wilbur, D. S., et al., 1997).

[0159] 8.1,2,10,11-octadeutero-3,6,9-trioxa-1,11-undecanedinitrile (8) 1g (9.4 mmol) of diethylene glycol was dissolved in 2 mL D2O in a 10 mLround bottom flask under argon atmosphere. The D₂O was removed by rotaryevaporation and the process was repeated 4 times. The d-2 diethyleneglycol was additioned with 25 mL dry benzene, followed by 1.6 g (28.2mmol) d-3 acrylonitrile with stirring under argon atmosphere. After 12h, the solvent was removed under reduced pressure and the resultantsemisolid was extracted with chloroform (2×5 mL). The solvent wasremoved by rotary evaporation to yield 1.85 g (89%) product. (Ashikaga,K., et al., 1988).

[0160] 9. 2,3,11,12-octadeutero-4,7,10-trioxa-1,13-tridecanediamine (9)Raney nickel (Aldrich) was washed five times with anhydrous methanol byinversion and decantation. 50 mg of the washed catalyst was placed in 20mL anhydrous methanol, followed by 1 g (4.6 mmol) 8 in a 50 mL screw-capvial fitted with a Teflon-lined rubber septum. The vial headspace wasflushed for a few min with H₂ gas via an 18-gauge needle piercing theseptum. The cap was screwed on tightly and the entire assembly wascharged to 40 psi H₂ and placed in a hot water bath (80° C.) for 4hours, after which the solid catalyst was removed by filtration and themethanol evaporated. The final product was purified by reverse-phaseHPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobile phase: H₂O (0.08%TFA)/ACN (0.08% TFA)). Yield 180 mg. (Ashikaga, K., et al., 1988).

[0161] 10. Deuterated analog of 6 (10) 25 mg (0.09 mmol) 5 was added toa stirred solution of 90 mg (0.4 mmol) 9 in 5 mL 0.2M sodium carbonate,pH 10.5 at 37° C. The reaction was allowed to proceed for 3 days, afterwhich the solution was neutralized with dilute trifluoroacetic acid andpurified by reverse-phase HPLC (Vydac C-18 prep-scale column, 6 mL/min.Mobile phase: H₂O (0.08% TFA)/ACN (0.08% TFA)). Yield 6 mg.

[0162] 11. Deuterated analog of 7 (11) A 3 mg (8.4 mcmol) quantity of 4was dissolved in 0.7 mL anhydrous DMF with stirring, under argonatmosphere. 5 mcL triethylamine was added, followed by 2.4 mg (8.9mcmol) 1. The formation of active ester was monitored by silica TLC (5:1CHCl₃/CH₃OH, Rf 0.5, UV) by briefly drying the spotted TLC plate with astream of air. After 25 minutes, the mixture was added to 6 mg (11mcmol) 10 in 1 mL anhydrous DMF. After 2 hours, the solvent was removedby vacuum centrifugation and the final product was purified byreverse-phase HPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobilephase: H₂O (0.08% TFA)/ACN (0.08% TFA)). Yield 1.8 mg.

[0163] 12. GM1 internal standard conjugate (12) 1.8 mg 11 was added to 2mL of 100 mM Tris/10 mM MgCl2, pH 7.3 buffer with stirring. 15 unitsrecombinant β-D-galactosidase (Sigma) was added, and after 12 hours themixture was purified by reverse-phase HPLC (Vydac C-18 prep-scalecolumn, 6 mL/min. Mobile phase: H₂O (0.08% TFA)/ACN (0.08% TFA)). Yield1.5 mg.

[0164] Polyether Diamine Linker Synthesis (Second Generation)

[0165] Synthesis is based on chemistry previously described (Kataky, R.et. al., 1990), with minor modifications and an additional two steps. Asan example, deviations from the established procedure as well as exactdetails for the additional steps are outlined below for the startingmaterial diethylene glycol.

[0166] 1,11-Dicyano-3,6,9-trioxaundecane (13) To a stirred solution of2% (w/v) sodium hydroxide (5 mL) and diethylene glycol (5.3 g, 50 mmol)was added acrylonitrile (7.95 g, 150 mmol). The mixture was stirred atroom temperature overnight and additioned with 50 mL dichloromethane.The organic layer was washed 2× with brine and dried (MgSO₄). Thesolvent was removed by rotary evaporation. The oily residue was treatedwith 200 proof ethanol, and the solvent was removed by rotaryevaporation. This was repeated 2× to remove excess unreactedacrylonitrile. The product was used without further purification

[0167] Diethyl 4,7,10-trioxatridecane-1,13-dioate (14) 2 g (9.4 mmol)13, was dissolved in 5 mL ethanol. 1 g conc. sulfuric acid was addedslowly, over 5 minutes. The reaction was heated to reflux overnight. Thereaction was extracted with 40 mL methylene chloride, washed once with10 mL water and 3× with 10 mL dilute brine solution. The organic layerwas dried (MgSO₄) and solvent was removed to yield an oil. The finalproduct was purified by silica chromatography (methylene chloride/ethylacetate).

[0168] 1,13-dihydroxy-4,7,10-trioxatridecane (15) Prepared exactly asdescribed, using tetrahydrofuran as solvent. (1.7 g, 5.5 mmol 14, 50 mLdistilled [CaH₂] THF, 0.66 g, 16.5 mmol lithium aluminum hydride). Onceaddition was complete, excess LAH was quenched with ethanol, and thesalts precipitated by dropwise addition of saturated sodium sulfatesolution until a white precipitate formed. The solvent was removed, theprecipitate washed 6×30 mL with THF and the combined organic extractswere evaporated to yield an oil. Final product is purified by silicachromatography (first with methylene chloride then with ethyl acetateand finally with acetone).

[0169] 1,13-dichloro-4,7,10-trioxatridecane (analog using P2) (16) 1.1g, (4.9 mmol) 15 was added to 1.15 g (14.6 mmol) distilled pyridine in30 mL dry benzene with stirring, followed by 1.8 g (14.6 mmol) thionylchloride. The mixture was heated to reflux for 6 hours. After cooling inan ice bath, 5 mL 3M HCl was added with vigorous stirring. The organiclayer was separated, washed 3× with a dilute brine solution, and dried(NaSO₄) to yield a yellowish oil. After washing and removal of solvent,the dichloride was used without further purification.

[0170] Additional Steps:

[0171] 1,13-dicyano-4,7,10-trioxatridecane) (17) To a stirred solutionof 0.78 g (15.5 mmol) sodium cyanide in 4 mL dimethyl sulfoxide at 80°C. was added 1 g (3.9 mmol) of 16. After 2 hours, the reaction wasadditioned with 10 mL of saturated sodium chloride solution, 5 mL ofwater, and 50 mL ethyl acetate. The organic layer was washed 3× with abrine solution as before, after which the organic layer was dried(Na₂SO₄) and the solvents removed. The final product was purified bysilica chromatography (methlyene chloride/ethyl acetate). ESI-MS:predicted, 240.1; observed, 241.1 (M+H+)⁺.

[0172] 1,15-diamino-5,8,11-trioxapentadecane (18) A stirred solution of50 mL dry THF containing 0.42 g (10.4 mmol) fresh LAH was heated togentle reflux under argon for 15 minutes. 0.5 g (2 mmol) 17 in 15 mL dryTHF was added dropwise over 20 minutes, maintaining a gentle reflux. Theunreacted LAH was quenched with ethanol, and the mixture was treatedwith dropwise addition of saturated sodium sulfate under efficientstirring until a white precipitate formed. The mixture was filtered, andthe precipitate was washed 6×30 mL with THF. The organic extracts werecombined and the solvent was removed by rotary evaporation to yield anoil. ESI-MS: predicted, 248.1; observed, 249.1 (M+H+)⁺.

[0173] Deuteration

[0174] Deuterium has been incorporated into the diamine linker byreduction of 14 and 17 using lithium aluminum deuteride (98% D) toachieve a d-8 deuterated diamine. No other aspects of the synthesis werechanged for this procedure. These diols are used in the construction ofthe SFD conjugates as described later.

[0175] Clinical Substrate Synthesis for Sanfilippo Syndrome, type B(N-α-D-glucosaminidase deficiency).

[0176] 13. p-Aminophenyl-α-D-N-acetylglucosamine (19) 20 mg (0.07 mmol)p-Nitrophenyl-α-D-N-acetylglucosamine (Sigma) was added to 5 mg washedpalladium catalyst on activated carbon in 3 mL methanol with stirring ina 5 mL septa-lined vial. The septum was pierced by a 16-gauge needle andthe vial headspace was flushed with H₂ gas. H₂ gas was allowed to slowlybubble through the solution for 2 hours, after which the catalyst wasremoved by filtration over diatomaceous earth (Celite). The solvent wasremoved by rotary evaporation to yield a semi-solid 18 mg (90%).

[0177] 14. p-Acrylamidophenyl-α-D-N-acetylglucosamine (20) 10 mg (0.03mmol) 19 was added to 15 mL methanol and 100 mcL triethylamine withstirring. The solution was chilled in an ice bath. 15 mg (0.17 mmol)acryloyl chloride was dissolved in 2 mL dry methylene chloride and addeddropwise to the stirred solution over 5 minutes. The reaction wasallowed to return to room temperature, followed by 2 hours of stirring.The solution was then treated with successive anion and cation exchangeresins (AG MP-1 and AG MP-50, respectively, BioRad) until neutral pH wasobtained with moist pH paper. Solvent was removed by rotary evaporationto yield a solid (111 mg, 95%). 1H-NMR. Yield 11 mg.

[0178] 15. 3,6-dioxa-1,9-nonanedinitrile (21) 2 g (0.032 mol) ethyleneglycol was added to 0.5 g dry potassium hydroxide in 30 mL dry benzene,followed by 5 g (0.096 mmol) acrylonitrile with stirring overnight atroom temperature. The reaction was filtered and the solvent was removedby rotary evaporation to yield an oil. Final product was purified bysilica chromatography (chloroform/methanol) to yield a colorless oil 3.2g (60%).

[0179] 16. 4,7-dioxa-1,10-decanediamine (22) Raney nickel (Aldrich) waswashed five times with anhydrous methanol by inversion and decantation.50 mg of the washed catalyst was placed in 20 mL anhydrous methanol,followed by 1 g (6 mmol) 21 in a 50 mL screw-cap vial fitted with aTeflon-lined rubber septum. The vial headspace was evacuated with H₂ gasvia an 16-gauge needle piercing the septum. The cap was screwed ontightly and the entire assembly was charged to 40 psi H₂ and placed in ahot water bath (80° C.) for 4 hours, after which the solid catalyst wasremoved by filtration and the methanol evaporated. The final product waspurified by reverse-phase HPLC (Vydac C-18 prep-scale column, 6 mL/min.Mobile phase: H₂O (0.08% TFA)/ACN (0.08% TFA)).

[0180] 17. Michael addition product of 20 and 22 (23) 5 mg (0.015 mmol)20 was added to a stirred solution of 13 mg (0.06 mmol) 22 in 5 mL 0.2Msodium carbonate, pH 10.5 at 37° C. The reaction was allowed to proceedfor 3 days, after which the solution was neutralized with dilutetrifluoroacetic acid and purified by reverse-phase HPLC (Vydac C-18prep-scale column, 6 mL/min. Mobile phase: H₂O (0.08% TFA)/ACN (0.08%TFA)). Yield 6 mg.

[0181] 18. SFB substrate conjugate of 4 and 23 (24) A 4 mg (0.013 mmol)quantity of 4 was dissolved in 1.5 mL anhydrous DMF with stirring, underargon atmosphere. 10 mcL dry triethylamine was added, followed by 4 mg(0.015 mmol) 1. The formation of active ester was monitored by silicaTLC (5:1 CHCl₃/CH₃₀H, Rf 0.5, UV) by briefly drying the spotted TLCplate with a stream of air. After 25 minutes, the mixture was added to 6mg (0.012 mmol) 23 in 1 mL anhydrous DMF. After 2 hours, the solvent wasremoved by vacuum centrifugation and the final product was purified byreverse-phase HPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobilephase: H₂O (0.08% TFA)/ACN (0.08% TFA)). Yield 4.2 mg.

[0182] 19. 1,9-tetradeutero-3,6-dioxa-1,9-nonanedinitrile (25) 0.5 g (8mmol) ethylene glycol was added to 0.1 g dry potassium hydroxide in 20mL acetonitrile, followed by 1.4 g (24 mmol) d-3 acrylonitrile withstirring overnight at room temperature. The reaction was filtered andthe solvent was removed by rotary evaporation to yield an oil. Finalproduct was purified by silica chromatography (chloroform/methanol) toyield a colorless oil 0.9 g (65%).

[0183] 20. 1,9-tetradeutero-3,6-dioxa-1,9-nonanediamine (26) Raneynickel (Aldrich) was washed five times with anhydrous methanol byinversion and decantation. 20 mg of the washed catalyst was placed in 30mL anhydrous methanol, followed by 0.5 g (3 mmol) 25 in a 50 mLscrew-cap vial fitted with a Teflon-lined rubber septum. The vialheadspace was evacuated with H₂ gas via an 18-gauge needle piercing theseptum. The cap was screwed on tightly and the entire assembly wascharged to 40 psi H₂ and placed in a hot water bath (80° C.) for 4hours, after which the solid catalyst was removed by filtration and themethanol evaporated. The final product was purified by reverse-phaseHPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobile phase: H₂O (0.08%TFA)/ACN (0.08% TFA)).

[0184] 21. Deuterated analog of 23 (27) 20 mg (0.07 mmol)p-acrylamidophenyl β-D-galactoside was added to a stirred solution of 90mg (0.4 mmol) 26 in 5 mL 0.2M sodium carbonate, pH 10.5 at 37° C. Thereaction was allowed to proceed for 3 days, after which the solution wasneutralized with dilute trifluoroacetic acid and purified byreverse-phase HPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobilephase: H₂O (0.08% TFA)/ACN (0.08% TFA)). Yield 2 mg.

[0185] 22. Deuterated analog of 24 (28) A 2 mg (6.3 mcmol) quantity of 4was dissolved in 1.5 mL anhydrous DMF with stirring, under argonatmosphere. 5 mcL triethylamine was added, followed by 2.1 mg (7.6mcmol) 1. The formation of active ester was monitored by silica TLC (5:1CHCl₃/CH₃OH, Rf 0.5, UV) by briefly drying the spotted TLC plate with astream of air. After 35 minutes, the mixture was added to 4 mg (7 mcmol)27 in 1 mL anhydrous DMF. After 2 hours, the solvent was removed byvacuum centrifugation and the final product was purified byreverse-phase HPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobilephase: H₂O (0.08% TFA)/ACN (0.08% TFA)). Yield 1.2 mg.

[0186] 23. SFB internal standard conjugate (29) 1.2 mg 28 was added to 2mL of 100 mM Tris/10 mM MgCl2, pH 7.3 bufferwith stirring. 15 unitsrecombinant β-D-galactosidase (Sigma) was added, and after 12 hours themixture was purified by reverse-phase HPLC (Vydac C-18 prep-scalecolumn, 6 mL/min. Mobile phase: H₂O (0.08% TFA)/ACN (0.08% TFA)). Yield0.7 mg.

[0187] Clinical Substrate Synthesis for Sanfilippo Syndrome, type D (asulfatase deficiency).

[0188] 24. p-Acrylamidophenyl-α-D-N-acetylglucosamine-6-sulfate (30) 100mg (0.28 mmol) 20 was added to 10 mL dry DMF under argon atmosphere withstirring at room temperature. 89 mg (0.56 mmol) sulfur trioxide-pyridinecomplex was dissolved in 2 mL dry DMF and was added to the reaction in0.7×, 1.1×, 1.3× and 1.9× equivalents (+700 mcL, +400 mcL, +200 mcL, and+600 mcL). The reaction progress was monitored by 1H-NMR shift of theanomeric (C1) proton chemical shift from 5.29 to 5.24 ppm by removal of15 mcL of solution 1 hour after addition of each amount of sulfatingreagent. The removed mixture was dried by vacuum centrifugation andredissolved in d-6 DMSO and analyzed. Upon the appearance of more thantwo forms (starting material and C-6 sulfate) of the C1 anomeric proton,the reaction was removed to −20° C. and stored. The product was purifiedby vacuum centrifugation to remove solvent, followed by reverse-phaseHPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobile phase: H₂O (0.08%TFA)/ACN (0.08% TFA)). Yield 72%.

[0189] 25. Michael addition product of 18 and 30 (31) 25 mg (0.058mmol)30 was added to a stirred solution of 83 mg (0.35 mmol) 18 in 5 mL0.2M sodium carbonate, pH 10.5 at 37° C. The reaction was allowed toproceed for 3 days, after which the solution was neutralized with dilutetrifluoroacetic acid and purified by reverse-phase HPLC (Vydac C-18prep-scale column, 6 mL/min. Mobile phase: H₂O (0.08% TFA)/ACN(0.08%TFA)). Yield 10 mg.

[0190] 26. SFD substrate conjugate of 4 and 31 (32) A 5.7 mg (0.018mmol) quantity of 4 was dissolved in 1.0 mL anhydrous DMF with stirring,under argon atmosphere. 20 mcL dry triethylamine was added, followed by5.5 mg (0.020 mmol) 1. The formation of active ester was monitored bysilica TLC (5:1 CHCl₃/CH₃OH, Rf 0.5, UV) by briefly drying the spottedTLC plate with a stream of air. After 25 minutes, the mixture was addedto 10 mg (0.015 mmol) 31 in 1 mL anhydrous DMF. After 2 hours, thesolvent was removed by vacuum centrifugation and the final product waspurified by reverse-phase HPLC (Vydac C-18 prep-scale column, 6 mL/min.Mobile phase: H₂O (0.08% TFA)/ACN (0.08% TFA)). Yield 5.4 mg.

[0191] 27. 1,2,14,15-octadeutero-1,15-diamino-5,8,11-trioxapentadecane(33) as referenced in Polyether Diamine Linker Synthesis, SecondGeneration.

[0192] 28. Deuterated analog of 31 (34) 25 mg (0.07 mmol) 20 was addedto a stirred solution of 100 mg (0.4 mmol) 11 in 5 mL 0.2M sodiumcarbonate, pH 10.5 at 37° C. The reaction was allowed to proceed for 3days, after which the solution was neutralized with dilutetrifluoroacetic acid and purified by reverse-phase HPLC (Vydac C-18prep-scale column, 6 mL/min. Mobile phase: H₂O (0.08% TFA)/.ACN (0.08%TFA)). Yield 7 mg.

[0193] 29. SFD internal standard conjugate (35) A 4 mg (12.6 mcmol)quantity of 4 was dissolved in 1 mL anhydrous DMF with stirring, underargon atmosphere. 20 mcL triethylamine was added, followed by 4 mg (14mcmol) 1. The formation of active ester was monitored by silica TLC (5:1CHCl₃/CH₃OH, Rf 0.5, UV) by briefly drying the spotted TLC plate with astream of air. After 20 minutes, the mixture was added to 7 mg (11mcmol) 34 in 1 mL anhydrous DMF. After 4 hours, the solvent was removedby vacuum centrifugation and the final product was purified byreverse-phase HPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobilephase: H₂O (0.08% TFA)/ACN (0.08% TFA)). Yield 2. 7 mg.

[0194] N-(d-Biotinyl-sarcosinyl)-12-aminododecanoic acid (36). Compound4 (32.2 mg, 0.102 mmole) was dried overnight in vacuo (with P₂O₅). DryDMF (2 mL) was added and the mixture was stirred with warming to affectdissolution under nitrogen.

[0195] Triethylamine (34 mcL) was added followed by 1 (20.4 mcL, 0.115mmole) added in two 10.2 mcL portions, 5 min apart. The mixture wasstirred for 1 hr at room temperature under nitrogen. 12-Aminododecanicacid (24.1 mg, 0.112 mmole, Sigma) was added in one portion, and themixture was stirred at room temperature for 2 hr under nitrogen. CHCl₃(80 mL) was added, and the organic solution was washed with two 10 mLportions of 1 M HCl. CHCl₃ was removed by rotary evaporation, andresidual DMF was removed by vacuum centrifugation. The compound wasdissolved in methanol and purified by HPLC (Vydac 218TP prep column).Solvent program is: 0-10 min, water with 0.06% TFA; 10-55 min, 0-100%methanol with 0.06% TFA, flow rate is 6 mL/min. Yield 31.7 mg. 1H-NMR.ESI-MS, calculated 513.4, observed 513.4 (M+H)⁺

[0196] N-hydroxysuccinimidyl ester of 36 (37). Compound 36 (9.8 mg, 19mcmole) is dissolved in 100 mcL of dry DMF under nitrogen.N-hydroxysuccinimide (2.2 mg, 19 mcmole) was added followed bydicyclohexylcarbodiimide (3.9 mg, 19 mcmole). The mixture was stirred atroom temperature for 60 h in the dark. Solvent was removed by vacuumcentrifugation, and the residue was submitted to flash chromatography onsilica gel using a gradient of CHCl₃/CH₃OH (15/1) to CHCl₃/CH₃OH (12/1).Yield 9.8 mg. 1H-NMR. ESI-MS, calculated 610.8, observed 609.7 (M+H)⁺.

[0197] N-(N-(d-Biotinyl-sarcosinyl)-12-aminododecanoyl)-pyschosine (38).Compound 37 (6.2 mg, 10 mcmole) and pyschosine (4.7 mg, 10 mcmole,Sigma) were dissolved in 200 mcL of dry DMF under nitrogen.Diisoproylethylamine (5 mcL) was added, and the mixture was stirredunder nitrogen for 2 days in the dark. The compound was injecteddirectly onto the HPLC column (Vydac 218TP semi-prep), and the columnwas developed at 2 mL/min with 0-20 min, water with 0.06% TFA, then20-80 min, 0-100% methanol with 0.06% TFA. Yield 3.8 mg. 1H-NMR. ESI-MS,calculated 957.3, observed 956.8 (M+H)⁺.

[0198]N-(N-(d-Biotinyl-sarcosinyl)-12-aminododecanoyl)-sphingosylphosphorylcholine(39). Sphingosylphosphorylcholine (4.0 mg, Sigma) was mixed with 1 mLdry DMF and solvent was removed by vacuum centrifugation. This wasrepeated two more times. The final dried residue weighed 2.5 mg (5.4mcmole). To this residue was added 3.3 mg of 37 (5.4 mcmole), 150 mcL ofdry DMF, and 2.5 mcL of diisoproylethylamine. The mixture was stirredunder nitrogen in the dark for 3 days. The compound was injecteddirectly onto the HPLC column (Vydac 218TP semi-prep), and the columnwas developed at 2 mL/min with 0-20 min, water with 0.06% TFA, then20-80 min, 0-100% methanol with 0.06% TFA. Yield 3.8 mg. 1H-NMR. ESI-MS,calculated 960.3, observed 958.7 (M+H)⁺.

[0199] Conjugate of d-biotin with 1,13-diamino-4,7,10-trioxatridecane(40). Compound 2 was reacted with 1,13-diamino-4,7,10-trioxatridecane(Fluka) essentially as described for the synthesis of 3. The product waspurified by HPLC (Vydac 218TP, semi-prep) using 0-100% methanol with0.06% TFA over 30 min at 1.5 mL/min.

[0200] Iodoacetylated 40 (41). Compound 40 was treated with 5equivalents of iodoacetic anhydride (Aldrich) in dry DMF with stirringunder nitrogen for 4 h at room temperature. The product was purified onHPLC as for 40. The structure was confirmed by ESI-MS.

[0201] Octadeuterated 41 (42). The title compound was prepared as forthe 40 using 9 instead of 1,13-diamino-4,7,10-trioxatridecane.

[0202] Octadeuterated 42 (43). The title compound was prepared from 42as for 41. The structure was confirmed by ESI-MS.

[0203] Exemplary MS^(N) Techniques and Instrumentation

[0204] An automated LC-MS/MS system for the identification of proteinsby their amino acid sequence has been developed. A schematicrepresentation is shown in FIG. 7 The system, which consists of anautosample, a capillary HPLC system connected on-line to an ESI triplequadrupole MS/MS instrument and a data system is operated in thefollowing way: Proteins (typically separated by 1D or 2D gelelectrophoresis) are cleaved with a specific protease, usually trypsin.the resulting cleavage fragments are placed in an autosampler. Every 37minutes the autosampler injects one sample into the HPLC system and thepeptides are separated by capillary reverse-phase chromatography. Asseparated peptides elute from the chromatography column, they areionized by the ESI process, enter the MS and the mass to charge ratio(m/z) is measured. Any peptide ion whose intensity exceeds apredetermined intensity threshold is automatically selected by theinstrument and collided in the collision cell with inert gas. Thesecollisions result in peptide fragmentation, primarily at the bonds ofthe peptide backbone (collision induced dissociation, CID). The massesof the CID fragments are measured and recorded in the data system. TheCID spectrum of a peptide contains sufficient information to identifythe protein by searching sequence databases with the uninterpreted MS/MSspectra. This is accomplished with the Sequent program. The programidentifies each peptide in a sequence database which has the same massas the peptide that was selected in the MS for CID and predicts theMS/MS spectrum for each one of the isobaric peptides. By matching theexperimentally determined CID spectrum with computer generatedtheoretical CID spectra, the protein from which the observed peptideoriginated is identified. The system is capable of analyzing proteinsamples in a fully automated fashion at a pace of less than 40 min. persample. Since each peptide represents an independent proteinidentification and usually multiple peptides are derived from oneprotein, protein identification by this method is redundant and tolerantto proteins co-migrating in a gel. The system is well suited for thedetection and characterization of modified residues within polypeptidechains. The LC-MS/MS technique and automated analysis of the generatedCID spectra can be used for the methods of this invention.

[0205] Identification of Proteins at Sub-Femtomole Sensitivity bySolid-Phase Extraction Capillary Electrophoresis Tandem MassSpectrometry (SPE-CE-MSIMS)

[0206] Protein identification by this method is based on the sameprinciple as described above, except that peptide separation andionization are performed at significantly higher sensitivity. FIG. 8shows a schematic representation of the key design elements. The designof the system and its mode of operation have been published. Peptidesderived from protein digests are concentrated by SPE, separated by CEand analyzed by ESI-MS/MS. The resulting uninterpreted CID spectra areused to search sequence databases with the Sequest software system. TheSPE extraction device is a small reversed-phase chromatography column ofthe dimensions 0.18×1 mm which is directly packed in a fused silicaseparation capillary. Peptides contained in a sample solution areadsorbed and concentrated on the SPE device, eluted in an estimated100-300 nl of organic solvent and further concentrated byelectrophoretic stacking and/or isotachophoresis to an estimated volumeof 5-30 nl. The peptides are then separated by CE in a 20 μm or 50 μmi.d. capillary and directly ionized by ESI as they leave the capillary.With this system, peptide masses can be determined at a sensitivity of660 attomoles (approx. 500 fg for a 20 residue peptide) at aconcentration limit of 33 amol/μl and that proteins can be identified bythe CID spectra of automatically selected peptides at less than 10 fmol(0.5 ng for a protein of 50 kDa) of sample at a concentration limit ofless than 300 amol/μl. this technique is used for the analysis at veryhigh sensitivity of the peptide samples generated by the experiments. Ithas also been demonstrated that the analysis time available forautomated CID experiments can be significantly extended bydata-dependent modulation of the CE voltage. If several peptide ions aredetected coincidentally in the MS, the CE voltage is automaticallydropped. This results in a reduction of the electroosmotic flow out ofthe capillary and therefore in an extension of the time period availablefor selecting peptide ions for CID. The net effect of this peak parkingtechnique is an extension of the dynamic range of the technique becausethe increased time available is used for CID of ions with a low ioncurrent Once all the peptide ions are analyzed, electrophoresis isautomatically reaccelerated by increasing the CE voltage to the originalvalue. TABLE 1 Relative, redundant quantitation of α-lactalbuminabundance (after mixing with known amount of the same protein withcysteines modified with isotopically heavy biotinylated reagent) Peptidem/z Charge Ratio # (light) state Peptide Sequence (heavy:light) 1 518.42+ (K) IWCK 2.70 2 568.4 2+ (K) ALCSEK (SEQ ID NO:2) 2.68 3 570.4 2+ (K)CEVFR (SEQ ID NO:3) 2.90 4 760.5 2+ (K) LDQWLCEK (SEQ ID NO:4) 2.82 5710.1 3+ (K) FLDDDLTDDIMCVK (SEQ ID NO:5) 2.88 6 954.2 3+ (K)DDQNPHSSNICNISCDK (SEQ ID 2.90 NO:6) 7 1286.9 4+ (K)GYGGVSLPEWVCTTFHTSGYDT NA^(a) QAIVQNNDSTEYGLFQINNK (SEQ ID NO:7) (SEQ IDNO.

[0207] TABLE 2 Sequence identification and quantitation of thecomponents of a protein micture in a single analysis. Abserved Expectedratio ratio % Gene Name* Peptide sequence identified (d0/d8)^(†) Mean ±SD (d0/d8)⁼ error LCA_BOVIN ALC#SEK (SEQ ID NO:8 0.94 C#EVFR 1.03 0.96 ±0.06 1.00 4.2 FLDDLTDDIMC#VK (SEQ 0.92 ID NO:9) OVAL_CHICK ADHPFLFC#IK(SEQ ID 1.88 NO:10) 1.92 ± 0.06 2.00 4.0 YPILPEYLQC#VK (SEQ ID 1.96NO:11) BGAL_ECOLI LTAAC#FDR (SEQ ID 1.00 NO:12) 0.91 0.98 ± 0.07 1.002.0 IGLNC#QLAQVAER (SEQ ID NO:13) 1.04 IIFDGVNSAFHLWC#NGR (SEQ ID NO:14)LACB_BOVIN WENGEC#AQK (SEQ ID 3.64 NO:15) 3.55 ± 0.13 4.00 11.3LSFNPTQLEEQC#HI (SEQ 3.45 ID NO:16) G3P_RABIT VPTPNVSVVDLTC#R 0.54 (SEQID NO:17) 0.56 ± 0.02 0.50 12.0 IVSNASC#TTNC#LAPLAK 0.57 (SEQ ID NO:18)PHS2_RABIT IC#GGWQMEEADDWLR 0.32 (SEQ ID NO:19) 0.32 ± 0.03 0.33 3.1TC#AYTNHTVLPEALER 0.35 (SEQ ID NO:20) WLVLC#NPGLAEIIAER 0.30 (SEQ IDNO:21)

[0208] TABLE 3 Protein profiles from yeast growing on galactose orethanol as a carbon source. Observed Gene ratio^(†) Galactose- Glucose-name* Peptide sequence identified (Eth:Gal)⁼ repressed^(§) repressed^(§)ACH1 KHNC#LHEPHMLK (SEQ ID NO:22) >100:1 ✓ ADH1 YSGVC#HTDLHAWHGDWPLPVK(SEQ 0.57:1 ID NO: 23) 0.48:1 C#C#SDVFNQVVK (SEQ ID NO:24) ADH2YSGVC#HTDLHAWHGDWPLPTK (SEQ >200:1 ✓ ✓ ID NO:25) >200:1 C#SSDVFNHVVK(SEQ ID NO:26) ALD4 TFEVINPSTEEEIC#HIYEGR (SEQ ID >100:1 ✓ ✓ NO:27) BMH1SEHQVELIC#SYR (SEQ ID NO:28) 0.95:1 CDC19 YRPNC#PIILVTR (SEQ ID NO:29)0.49:1 NC#TPKPTSTTETVAASAVAAVFEQK 0.65:1 (SEQ ID NO:30) 0.67:1 AC#DDKFBA1 SIAPAYGIPVVLHSDHC#AK (SEQ ID 0.60:1 NO:31) 0.63:1 EQVGC#K (SEQ IDNO:32) GAL1 LTGAGWGGC#TVHLVPGGPNGNIEK 1:>200 ✓ (SEQ ID NO:33) GAL10HHIPFYEVDLC#DR (SEQ ID NO:34) 1:>200 ✓ DC#VTLK (SEQ ID NO:35) 1:>200GCY1 LWC#TQHHEPEVALDQSLK (SEQ ID 0.34:1 ✓ NO:36) GLK1IC#SVNLHGDHTFSMEQMK (SEQ ID 0.65:1 NO:37) GPD1 IC#SQLK (SEQ ID NO:38)0.54:1 ✓ ICL1 GGTQC#SIMR (SEQ ID NO:39) >100:1 ✓ IPP1NC#FPHHGYIHNYGAFPQTWEDPNVS- 0.76:1 HPETK (SEQ ID NO:40) LPD1VC#HAHPTLSEAFK (SEQ ID NO:41) 1.30:1 ✓ PEP4 KGWTGQYTLDC#NTR (SEQ IDNO:42) 2.60:1 ✓ PSA1 SVVLC#NSTIK (SEQ ID NO:43) 0.56:1 PGM2C#TGGIILTASHNPGGPENDMGIK (SEQ 0.58:1 ✓ ID NO:44) 0.62:1LSIC#GEESFGTGSNHVR (SEQ ID NO:45) PCK1 C#PLK 1.59:1 IPC#LADSHPK (SEQ IDNO:46) 1.47:1 C#INLSAEKEPEIFDAIK (SEQ ID NO:47) 1.52:1 ✓ C#AYPIDYIPSAK(SEQ ID NO:48) 1.41:1 IVEEPTSKDEIWWGPVNKPC#SER 1.85:1 (SEQ ID NO:49)QCR6 ALVHHYEEC#AER (SEQ ID NO:50) 1.30:1 ✓ RPL1A^(¶) SC#GVDAMSVDDLKK(SEQ ID NO:51) 0.82:1 SAH1 HPEMLEDC#FGLSEETTTGVHHLYR 0.62:1 (SEQ IDNO:52) 0.74:1 EC#INIKPQVDR (SEQ ID NO:53) SOD1GFHIHEFGDATNGC#VSAGPHFNPFK 0.46:1 ✓ (SEQ ID NO:54) TEF1 RGNVC#GDAK (SEQID NO:55) 0.81:1 C#GGIDK (SEQ ID NO:56) 0.70:1 FVPSKPMC#VEAFSEYPPLGR(SEQ ID 0.74:1 NO:57) VMA2 IPIFSASGLPHNEIAAQIC#R (SEQ ID 0.70:1 NO:58)YHB1 HYSLC#SASTK (SEQ ID NO:59) 0.69:1

[0209] TABLE 4 Disease Enzyme Dysfunction Butyrylcholinesterasedeficiency BCHE Decreassed or absent enzyme activity Essentialfructosuria hepatic Fuctokinase Deficient enzyme activity fructokinasedeficiency Hereditary fructose intolerance Fructose 1,6- Deficientenzyme activity diphosphate deficiency bisphosphatase Erythrocytealdolase deficiency Fructose 1,6- Deficient enzyme activity withnonspherocytic hemolytic biphosphatase aldolase A anemia (aldolase Adeficiency) Glycogen storage disease type Glucose 6-phosphatase Absentor deficient Ia (von Gierke disease) enzyme activity Glycogen storagedisease type Glucose 6-phosphate Deficient transport of Ib tranlocaseglucose 6-phosphate across the membrane of endoplasmic reticulumGlycogen storage disease type Amylo-1, 6-glucosidase Absent or deficientIII (debrancher enzyme) enzyme activity Glycogen storage disease typeα-1, 4 glucan-6-α- Deficient enzyme activity IV (Andersen disease)glucosyltransferase Glycogen storage disease type Muscle glycogen Absentor deficient V (McArdle disease) phosphorylase enzyme Glycogen storagedisease X- Phosporylase b-kinase Deficient or absent linkedphosphorylase kinase enzyme activity function deficiency Glycogenstorage disease Phosphorylase b-kinase Deficient enzyme activityautosomal phosphorylase kinase deficiency Glycogen storage disease liverLiver phosphorylase Deficient enzyme activity phosphorylase deficiencyGlycogen storage disease type Muscle Deficient enzyme activity VII(Tarui disease) phosphofructokinase 1 Liver glycogen synthase Liverglycogen synthase Unknown deficiency Phosphoglycerate kinasePhosphoglycerate kinase Deficient enzyme deficiency Phosphoglyceratemutase Phosphoglycerate Deficient enzyme deficiency mutase Musclelactate dehydrogenase Muscle-specific subunit of Absence of M subunit ofdeficiency lactate dehydrogenase LDH. Muscle LDH is a (LDH) tetramer ofthe heart- specific subunit Glucose phosphate isomerase Glucosephosphate Unknown deficiency isomerase Transferase deficiency Galactose1-phosphate Deficient enzyme activity galactosemia uridyltransferaseGalactokinase deficiency Galactokinase Deficient enzyme activitygalactosemia Epimerase deficiency Uridine diphosphate Deficient enzymeaction galactosemia galactose-4-epimerase in blood cells only (benign)or, more rarely, in all tissues (generalized) Phenylketonuria (PKU) dueto Phenylalanine Deficient or absent PAH PAH deficiency hydroxylase(PAH) activity (<1% normal) Hyperphenylalaninemia due toDihydropteridine Deficient or absent DHPR DHPR-deficiency reductase(DHPR) activity Hyperphenylalanineemia due to Guanosine triphosphateDeficient enzyme activity GTP-CH-deficiency cyclohydrolase (GTP-CH)Hyperphenylalaninemia due to 6-Pyruvoyl Deficient enzyme activity6-PTS-deficiency tetrahydropterin synthase (6-PTS) Oculocutaneoustyrosinemia Tyrosine Decreased activity (tyrosinemia type II; tyrosineaminotransferase amino-transferase deficiency) 4-Hydroxyphenylpyruvicacid 4-Hydroxy-phenylpyruvic Decreased activity dioxygenase (tyrosinemiatype acid dioxygenase III) Maleylacetoacetate isomeraseMaleylacetonacetate Presumably decreased deficiency (tyrosinemia typeIb) isomerase enzyme activity (tentative) Hepatorenal tyrosinemiaFumarylaceoacetate Deficient enzyme activity (tyrosinemia type I:hydroxylase fumarylacetoacetate hydrolase deficiency) Carbamyl phosphatesynthetase Carbamyl phosphate Absent or deficient deficiency synthetaseI enzyme activity Orinthine transcarbamylase Orinthine Absent or reduceddeficiency transcarbamylase enzyme activity Argininosuccinic acidArgininosuccinic acid Deficient enzyme activity synthetase deficiencysynthetase Argininosuccinase deficiency Argininosuccinate lyaseDeficient enzyme activty Arginase deficiency Liver arginase Deficientenzyme activity Familial hyperlysinemia α-Aminoadipic Deficient enzymeactivity (variant: saccharopinuria) semialdehyde synthase Maple syrupurine disease Branched-chain α-keto Deficient or absent (<2%) (MSUD) orbranched chain acid dehydrogenase BCKAD complex activity ketoacidemia inmitochondria; immunologic absence or reduced levels of enzyme subunits;impairment of E1 subunit assembly Cystathionine β-synthase Cystathionineβ-synthase Deficient enzyme activity deficiency α-Cystathionasedeficiency α-Cystathionase Deficient enzyme activity Hepatic methionineIsoenzyme of methionine Deficient enzyme activity adenosyltransferasedeficiency adenosyltransferase Sarcosinemia Sarcosine Deficient enzymeactivity dehydrogenase? Nonketonic hyperglycinemia Glycine cleavagesystem Deficient enzyme activity Hyperuracil thyminuriaDihydropyrimidine Deficient enzyme activity dehydrogenaseDihydropyrimidinuria Dihydropyrimidinase Unknown Pyridoxine dependencywith Brain glutamic acid Deficient coenzyme seizures decarboxylase-1binding? (brain) GABA aminotransferase GABA-α-ketoglutarate Deficientenzyme activity deficiency transaminase 4-Hydroxybutyric aciduriaSuccinc semialdehyde Deficient enzyme activity dehydrogenase Serumcarnosinase deficiency Serum carnosinase Deficient enzyme andhomocarnosinosis Alkaptonuria Homogentisic acid Absent or deficientoxidase enzyme activity Isovaleric acidemia Isovaleryl-CoA Deficientenzyme activity, dehydrogenase deficient protein, abnormal peptide sizeIsolated 3-methylcrotonyl-CoA 3-Methylcrotonyl-CoA Deficient enzymeactivity carboxylase deficiency carboxylase 3-Methylglutanconic aciduria3-Methylglutaconyl-CoA Deficient enzyme activity Mild form: hydratase3-Hydroxy-3-methylglutaryl- 3-Hydroxy-3- Deficient enzyme activity CoAlyase deficiency methylglutaryl-CoA lyase Mevalonic aciduria Mevalonatekinase Deficient enzyme activity Mitochondrial acetoacetyl-CoAMitochondrial Deficient enzyme activity, thiolase deficiencyacetoacetyl-CoA thiolase decreased protein, (T2) unstable proteinPropionic acidemia (2 nonallelic Propionyl-CoA Deficient enzyme activityforms designated pccA and carboxylase (PCC) (nonalleic forms reflectpccBC) mutations in nonidentical subunits of PCC) Methylmalonic acidemia(2 Methyldmalonyl-CoA Absent MUT activity in allelic variants designatedmut° mutase (MUT) mut°, deficient MUT and mut⁻ apoenzyme activity due toreduced affinity for cofactor (adenosylcobalamin) in mut⁻ Glutaricacidemia type I Glutaryl-CoA Deficient enzyme activity dehydrogenaseCytochrome oxidase deficiency Cytochrome oxidase Decreased activity ofthe polypeptides cytochrome oxidase complex Pyruvate dehydrogenasePyruvate decaroxylase, Decreased enzyme complex deficiency-E₁ E₁αactivity, decreased decarboxylase component protein Pyruvatedehydrogenase E₂ Dihydrolipoamide Decreased enzyme transacylasetransacylase activity; abnormal protein electrophoretic mobilityCombined α-ketoacid Lipoamide Decreased enzyme dehydrogenasedehydrogenase activity deficiency/lipoamide dehydrogenase deficiencyPyruvate carboxylase deficiency Pyruvate carboxylase Absent enzymeactivity; 7 cases absent enzyme, protein, and mRNA Carnitine palmitoyltransferase I Carnitine palmitoyl Deficient enzyme (CPT I) deficiencytransferase I Carnitine/acylcarnitine Carnitine/acylcarnitine Deficienttranslocase translocase deficiency translocase Carnitine palmitoyltransferase II Carnitine palmitoyl Deficient enzyme (CPT II) deficiencytransferase II Very long-chain acyl-CoA Very long-chain acyl-CoADeficient enzyme dehydrogenase (VLCAD) dehydrogenase deficiencyLong-chain acyl-CoA Long-chain acyl-CoA Deficient enzyme dehydrogenase(LCAD) dehydrogenase Long-chain L-3-hydroxyacyl- L-3-hydroxyacyl-CoADeficient enzyme CoA dehydrogenase (LCHAD) dehydrogenase deficiencyTrifunctional enzyme (TFE) Trifunctional enzyme Defi- deficiency cientenzyme Dienolyl-Co reductase 2,4-dienoyl-CoA Deficient enzyme deficiencyreductase Medium-chain acyl-CoA Medium-chain acyl-CoA Deficient enzymedehydrogenase (MCAD dehydrogenase deficiency Short-chain acyl-CoAShort-chain acyl-CoA Deficient enzyme dehydrogenase (SCAD) dehydrogenasedeficiency Glutaric acudemia type II Electron transfer In some cases, noflavoprotein (ETF); enzyme antigen; in ETF:ubiquinone others, no enzymeoxidoreductase activity Glycerol kinase deficiency Glycerol kinase Themicrodeletion (Gkd) involves not only GK but also the other deletedloci: AHC, DMD, OTC, and other lkinked loci Primary gout: superactivePP-ribose-P synthetase Enhanced enzyme variant of activityphosphoribosylpyrophosphate (PP-ribose-P) synthetase Primary gout:partial deficiency Hypoxanthine guanine Absent or deficient ofhypoxanthine guanine phosphoribosyl enzyme activityphosphoribosyltransferase transferase (HPRT) (HPRT) Lesch-Nyhansyndrome: Hypoxanthine guanine Absent or deficient deficiency ofhypoxanthine phosphoribosyltrans- enzyme activity guanine ferase (HPRT)phosphoribosyltransferase (HPRT) 2,8-Dihydroxyadenine lithiasis AdenineType I: absent enzyme (adenine phosphoribosyltransferase activity; typeII: reducted phosphoribosyltransferase affinity for PP-ribose-Pdeficiency) Adenosine deaminase Adenosine deaminase Absent or greatlydeficiency with severe combined diminished enzyme immunodeficiencydisease activity Purine nucleoside Purine nucleoside Absent or greatlyphosphorylase deficiency with phosphorylase diminshed enzyme cellularimmunodeficiency activity Myoadenylate deaminase Myoadenylate deaminaseNo enzyme activity; no deficiency (AMPDI) immunoreactive proteinXanthinuria Xanthine dehydrogenase Type I: absent xanthine (xanthineoxidase) dehydrogenase activity; type II: absent xanthine dehydrogenaseand aldehyde oxidase activity Hereditary orotic aciduria UMP synthaseDeficient enzyme activity (unstable protein) Pyrimidine 5′-nucleotidasePyrimidine 5′- Absent or unstable deficiency nucleotidase enzymeDihydropyrimidine Dihydropyrimidine Absent or unstable dehydrogenasedeficiency dehydrogenase enzyme Dihydropyrimidase deficiencyDihydropyrimidase Absent or unstable enzyme Familial lipoprotein lipaseLipoprotein lipase Nonfunctional protein in deficiency some, nondectableenzyme activity and protein in others Familial lecithin: cholesterolLecithin:cholesterol Absent enzyme protein or acyltransferase deficiencyacyltransferase deficient enzyme activity δ-Aminolevulinic acidδ-Aminolevulinic acid Minimal enzyme activity dehydratase porphyriadehydratase Acute intermittent porphyria Porphobilinogen Decreasedenzyme deaminase activity (˜50%) Congenital erythropoieticUroporphyrinogen III Minimal enzyme activity porphyria cosynthasePorphyria cutanea tarda (familial Uroporphyrinogen Decreased enzymeform) decarboxylase activity (˜50%) Hepatoerythropoietic porphyriaUroporphyrinogen Minimal enzyme activity decarboxylase Hereditarycoproporphyria Coproporphyrinogen Decreased enzyme oxidase activity(˜50%) Variegate porphyria Protoporphyrinogen Decreased enzyme oxidaseactivity (˜50%) Erythropoietic protophorphyria Ferrochelatase Decreaseenzyme activity (˜50%) Crigler-Najjar syndrome, type I Bilirubin UDP-Absent enzyme activity glucuronsyltransferase Crigler-Najjar syndrome,type II Billirubin UDP- Markedly reduced glucuronosyltransferase, enzymeactivity Gilbert syndrome Bilirubin UDP- Reduced enzyme activityglucuronosyltransferase activity Refsum disease Phytanic acid α-Deficient enzyme activity hydroxylase Primary hyperoxaluria type 1Alanine-glyoxylate Loss of enzyme catalytic aminotransferase activityand aberrant subcellular distribution Primary hyperoxalauria type 2Glyoxylate reductase/D- Loss of enzyme catalytic glycerate dehydrogenaseactivity G_(M2) gangliosidosis: β-hexosaminidase Absent or defectivehexosamindase α-subunit hexosaminidase A (αβ) deficiency (variant B,Tay-Sachs activity disease) Glycogen storage disease type α-glucosidaseAbsent or deficient II enzyme activity Mucopolysaccharidosis Iα-L-iduronidase Absent enzyme activity (Hurler, Scheie, and Hurler-Sheie syndromes, MPS, MPS IS, MPS IH/S) Mucopolysaccharidosis IIIduronate sulfutase Absent enzyme activity (Hunter syndrome)Mucopolysaccharidosis III IIIA: Heparan N-sulfatase Absent enzymeactivity (Saniflippo syndrome) types A, IIIB: α-N-acetyl- B, C and Dglucosaminidase IIC: Acetyl-CoA: α- glucosaminidine acetyltransferaseIIID: N-acetyl- glucosamine-6-sulfatase Mucopolysaccharidosis IV IVA:Galactose 6- Absent enzyme activity (Morquio syndrome) types A sulfataseand B IVB: β-Galactosidase Mucopolysaccharidosis VIN-acetyl-galactosamine Absent enzyme activity (Maroteaux-Lamy syndrome)4-sulfatase Mucopolysaccharidosis VII (Sly β-glycuronidase Absent enzymeactivity syndrome) I-cell disease (ML-II) N-acetylglucosaminyl-l-Phorphorylation of many phosphotransferase lysosomal enzymes Schindlerdisease (α-N-acetyl- α-N-acetyl- Deficient activity of α-N-galactosaminidase deficiency) galactoaminidase acetylgalactosaminidaseα-Mannosidosis α-D-mannosidase Deficient or unstable enzyme activityβ-Mannosidosis β-D-mannosidase Deficient enzyme activity Sialidosisα-neuraminidase Deficient enzyme activity AspartylglucosaminuriaAspartyglucosaminidase Deficient enzyme activity Fucosidosisα-L-fucosidase Deficient enzyme activity Wolman disease and cholestrylAcid lipase Deficient enzyme activity ester storage disease Ceramidasedeficiency (Farber Ceramidase Deficient enzyme activitylipogenulomatosis) Nielmann-Pick disease (NPD) SphingomyelinaseDeficient types A and B (primary sphingomyelinase activity sphingomyelinstorage) Gaucher disease type I Glucoserebrosidase Decreased catalytic(nonneuronopathic) activity and some instability of enzyme proteinGloboid-cell leukodystrophy Galactosylceramidase Absent enzyme activity(Krabbe disease) Metachromatic leukodystrophy Arylsulfatase A Deficientenzyme activity Fabry disease α-Galactosidase A Nonfunctional orunstable enzyme protein G_(M1) gangliosidosis Acid β-galactosidaseDeficient enzyme activity (GLBI) G_(M2) gangliosidosis: β-hexosaminidaseAbsent or defective hexosaminidase α-subunit hexosaminidase A (αβ)deficiency (variant B, Tay-Sachs activity disease) Steroid21-hydroxylase Steroid 21-hydroxylase Absent or truncated deficiencysalt-losing form enzyme with no activity Steroid 5α-reductase 2 Steroid5α-reductase 2 Absent or unstable deficiency enzyme activity Steroidsulfatase deficiency (X- 3β-hydroxysteroid Absent immunoreactive linkedichthyosis) sulfatase and enzymatically active protein (both deletionand nondeletion patients) Methylenetetrahydrofolate Methylenetetrahydro-Absent or deficient reductase deficiency folate reductase enzymeactivity. Thermolabile variants have been described. Holocarboxylasesynthetase Holocarboxylase Deficient holocarboxylase deficiencysynthetase synthetase activity Biotinidase deficiency BiotinidaseDeficient biotinidase activity Hereditary methmoglobinemia Cytochrome b₅reductase Deficient enzyme activity secondary to cytochrome b₅ inerthrocyte cytosol only reductase deficiency, types I, II, (type I), inall tissues and III (type II), and in all hematopoietic cells (type III)Pyruvate kinase deficiency Pyruvate kinase Deficient enzyme activityhemolytic anemia Hexokinase deficiency Heokinase Deficient enzymeactivity hemolytic anemia Glucosephosphate isomerase GlucosephosphateDeficient enzyme activity deficiency hemolytic anemia isomerase Aldolasedeficiency hemolytic Aldolase (A type) Deficient enzyme activity anemiaTriosephosphate isomerase Triophosphate Enzyme activity deficientdeficiency hemolytic anemia isomerase in all tissues Phosphoglyceratekinase Phosophoglycerate kinase Deficient enzyme activity deficiencyhemolytic anemia in hemizygotes 2,3-Diphospho-glyceromutase2,3-Diphospho-glycerate- Deficienct enzyme activity and phosphatasedeficiency mutase and phosphatase (1 protein) 6-Phosphogluconate6-Phosphogluconate Enzyme activity dehydrogenase deficiencydehydrogenase deficiency Glutathione peroxidase Glutathione perocidaseDiminshed enzyme deficiency activity Glutathione reductase Glutathionereductase Deficient enzyme activity deficiency Glutathione synthetaseGlutathione synthetase Deficient enzyme activity deficiency hemolyticanemia y-Glutamylcysteine synthetase y-Glutamylcysteine Deficient enzymeactivity deficiency hemolytic anemia synthetase Adenosine deaminaseAdenosine deaminase Overproduction of hyperactivity hemolytic anemiastructurally normal enzyme protein mediated at mRNA translation levelPyrimidine nucleotidase Pyrimidine nucleotidase Deficient enzymeactivity deficiency hemolytic anemia Myleoperoxidase deficiencyMyeloperoxidase Absent or deficient enzyme activity Carbonic anhydraseII Carbonic anhydrase II Quantitative deficiency of deficiency syndromecarbonic anhydrase II (osteopetrosis with real tubular acidosis)Albinism, oculocutaneous Tyrosinase Absent, reduced, ortyrosinase-negative type unusual enzyme activity (OCAIA) Canavan diseaseAspartoacylase Deficient enzyme activity

[0210] TABLE 5 Molecular masses of protonated and sodiatedsubstrate-conjugates, products, and internal standards for CDGS enzymes.m/z Internal Substrate Product standard Enzyme (M + H)⁺ (M + Na)⁺ (M +H)⁺ (M + Na)² (M + H)⁺ (M + Na)⁺ Type Ia,b 711 733   549   571   555  577 Manose- 725 747   563   585   570   592 transferase Type II 11561178  1343  1365  1348  1370 Type 1126 1148 2362^(a) 2384^(a) 2367^(a)2389^(a)

[0211]

REFERENCES

[0212] Ashikaga, K. et al. (1988) Bull. Chem. Soc. Jpn. 61:2443-2450.

[0213] Bayer, E. and Wilchek, M. (eds.) “Avidin=Biotin Technology,”(1990) Methods Enzymol. 184:49-51.

[0214] Bleasby, A. J. et al. (1994), “OWL—a non-redundant compositeprotein sequence database,” Nucl. Acids Res. 22:3574-3577.

[0215] Boucherie, H. et al. (1996), “Two-dimensional gel proteindatabase of Saccharomyces cerevisiae,” Electrophoresis 17:1683-1699.

[0216] Brockhausen, I.; Hull, E.; Hindsgaul, O.; Schachter, H.; Shah, R.N.; Michnick, S. W.; Carver, J. P. (1989) Control of glycoproteinsynthesis. J. Biol. Chem. 264, 11211-11221.

[0217] Chapman, A.; Fujimoto, K.; Kornefeld, S. (1980) The primaryglycosylation defect in class E Thy-1-negative mutant mouse lymphomacells is an inability to synthesize dolichol-P-mannose. J. Biol. Chem.255, 4441-4446.

[0218] Chen, Y.-T. and Burchell, A. (1995), The Metabolic and MolecularBases of Inherited Disease, Scriver, C. R. et al. (eds.) McGraw-Hill,New York, pp. 935-966.

[0219] Clauser, K. R. et al. (1995), “Rapid mass spectrometric peptidesequencing and mass matching for characterization of human melanomaproteins isolated by two-dimensional PAGE,” Proc. Natl. Acad. Sci. USA92:5072-5076.

[0220] Cole, R. B. (1997) Electrospray Ionization Mass Spectrometry:Fundamentals, Instrumentation and Practice, Wiley, New York.

[0221] De Leenheer, A. P. and Thienpont, L. M. (1992), “Application ofisotope dilution-mass spectrometry in clinical chemistry,pharmacokinetics, and toxicology,” Mass Spectrom. Rev. 11:249-307.

[0222] DeRisi, J. L. et al. (1997), “Exploring the metabolic and geneticcontrol of gene expression on a genomic scale,” Science 278:680-6

[0223] Dongr'e, A. R., Eng, J. K., and Yates, J. R., 3rd (1997),“Emerging tandem-mass-spectrometry techniques for the rapididentification of proteins,” Trends Biotechnol. 15:418-425.

[0224] Ducret, A., VanOostveen, I., Eng, J. K., Yates, J. R., andAebersold, R. (1998), “High throughput protein characterization byautomated reverse-phase chromatography/electrospray tandem massspectrometry,” Prot. Sci. 7:706-719.

[0225] Eng, J., McCormack, A., and Yates, J. I. (1994), “An approach tocorrelate tandem mass spectral data of peptides with amino acidsequences in a protein database,” J. Am. Soc. Mass Spectrom. 5:976-989.

[0226] Figeys, D. et al. (1998), “Electrophoresis combined with massspectrometry techniques: Powerful tools for the analysis of proteins andproteomes,” Electrophoresis 19:1811-1818.

[0227] Figeys, D., and Aebersold, R. (1998), “High sensitivity analysisof proteins and peptides by capillary electrophoresis tandem massspectrometry: Recent developments in technology and applications,”Electrophoresis 19:885-892.

[0228] Figeys, D., Ducret, A., Yates, J. R., and Aebersold, R. (1996),“Protein identification by solid phase microextraction-capillary zoneelectrophoresis-microelectrospray-tandem mass spectrometry,” NatureBiotech. 14:1579-1583.

[0229] Figeys, D., Ning, Y., and Aebersold, R. (1997), “Amicrofabricated device for rapid protein identification bymicroelectrospray ion trap mass spectrometry,” Anal. Chem. 69:3153-3160.

[0230] Freeze, H. H. (1998) Disorders in protein glycosylation andpotential therapy. J. Pediatrics 133, 593-600.

[0231] Freeze, H. H. (1999) Human glycosylation disorders and sugarsupplement therapy. Biochem. Biophys. Res. Commun. 255, 189-193.

[0232] Gamper, H. B., “Facile preparation of nuclease resistant3′-modified oligodeoxy-nucleotides,” Nucl. Acids Res., 21:145-150 (Jan1993)

[0233] Garrels, J. I., McLaughlin, C. S., Warner, J. R., Futcher, B.,Latter, G. I., Kobayashi, R., Schwender, B., Volpe, T., Anderson, D. S.,Mesquita, F.-R., and Payne, W. E. (1997), “Proteome studies ofSaccharomyces cerevisiae: identification and characterization ofabundant proteins. Electrophoresis,” 18:1347-1360.

[0234] Gerber, S. A.; Scott, C. R.; Turecek, F.; Gelb, M. H. (1999)Analysis of rates of multiple enzymes in cell lysates by electrosprayionization mass spectrometry. J. Am. Chem. Soc. 121, 1102-1103.

[0235] Glaser, L. (1966) Phosphomannomutase from yeast. In Meth.Enzymol. Vol. VIII, Neufeld, E. F.; Ginsburg, V. Eds; Academic Press:New York 1966, pp. 183-185.

[0236] Gygi, S. P. et al. (1999), “Correlation between portein and mRNAabundance in yeast,” Mol. Cell. Biol. 19:1720-1730.

[0237] Gygi, S. P. et al. (1999), “Protein analysis by mass spectrometryand sequence database searching: tools for cancer research in thepost-genomic era,” Electrophoresis 20:310-319.

[0238] Haynes, P. A., Fripp, N., and Aebersold, R. (1998),“Identification of gel-separated proteins by liquid chromatographyelectrospray tandem mass spectrometry: Comparison of methods and theirlimitations,” Electrophoresis 19:939-945.

[0239] Hodges, P. E. et al. (1999), “The Yeast Proteome Database (YPD):a model for the organization and presentation of genome-wide functionaldata,” Nucl. Acids Res. 27:69-73.

[0240] Johnston, M. and Carlson, M. (1992), in The Molecular andCellular Biology of the Yeast Saccharomyces, Johnes, E. W. et al.(eds.), Cold Spring Harbor Press, New York City, pp. 193-281.

[0241] Kataky, R. et. al. J Chem Soc Perk T 2 (2) 321-327 FEB 1990.

[0242] Kaur, K. J.; Hingsgaul, O. (1991) A simple synthesis of octyl3,6-di-O-(α-D-mannopyranosyl)-β-D-manopyranoside and its use as anacceptor for the assay of N-acetylglucosaminetransferase I activity.Glycoconjugate J. 8, 90-94.

[0243] Kaur, K. J.; Alton, G.; Hindsgaul, O. (1991) Use ofN-acetylglucosaminyltranserases I and II in the preparative synthesis ofoligosaccharides. Carbohydr. Res. 210, 145-153.

[0244] Korner, C.; Knauer, R.; Holzbach, U.; Hanefeld, F.; Lehle, L.;von Figura, K. (1998) Carbohydrate-deficient glycoprotein syndrome typeV: deficiency of dolichyl-P-Glc:Man9GlcNAc2-PP-dolichylglucosyltransferase. Proc Nati Acad Sci U.S.A. 95, 13200-13205.

[0245] Link, A. J., Hays, L. G., Carmack, E. B., and Yates, J. R., 3rd(1997), “Identifying the major proteome components of Haemophilusinfluenzae type-strain NCTC 8143,” Electrophoresis 18:1314-1334.

[0246] Link, J. et al. (1999), “Direct analysis of large proteincomplexes using mass spectrometry,” Nat. Biotech. 17:676-682 (July 1999)

[0247] Mann, M., and Wilm, M. (1994), “Error-tolerant identification ofpeptides in sequence databases by peptide sequence tags,” Anal. Chem.66:4390-4399.

[0248] McMurry, J. E.; Kocovsky, P. (1984) A method for thepalladium-catalyzed allylic oxidation of olefins. Tetrahedron Lett. 25,4187-4190.

[0249] Morris, A. A. M. and Turnbull, D. M. (1994) Curr. Opin. Neurol.7:535-541.

[0250] Neufeld, E. and Muenzer, J. (1995), “The mucopolysaccharidoses”In The Metabolic and Molecular Bases of Inherited Disease, Scriver, C.R. et al. (eds.) McGraw-Hill, New York, pp. 2465-2494.

[0251] Oda, Y. et al. (1999), “Accurate quantitation of proteinexpression and site-specific phosphorylation,” Proc. Natl. Acad. Sci.USA 96:6591-6596.

[0252] Okada, S. and O'Brien, J. S. (1968) Science 160:10002.

[0253] Opiteck, G. J. et al. (1997), “Comprehensive on-line LC/LC/MS ofproteins,” Anal. Chem. 69:1518-1524.

[0254] Paulsen, H.; Meinjohanns, E. (1992) Synthesis of modifiedoligosaccharides of N-glycoproteins intended for substrate specificitystudies of N-acetylglucosaminyltransferases II-V Tetrahedron Lett. 33,7327-7330.

[0255] Paulsen, H.; Meinjohanns, E.; Reck, F.; Brockhausen, I. (1993)Synthese von modifizierten Oligosacchariden der N-Glycoproteine zurUntersuchung der Spezifitat der N-Acetylglucosaminyltransferase II.Liebigs Ann. Chem. 721-735.

[0256] Pennington, S. R., Wilkins, M. R., Hochstrasser, D. F., and Dunn,M. J. (1997), “Proteome analysis: From protein characterization tobiological function,” Trends Cell Bio. 7:168-173.

[0257] Preiss, J. (1966) GDP-mannose pyrophosphorylase fromArthrobacter. In Meth. Enzymol. Vol. VIII, Neufeld, E. F.; Ginsburg, V.Eds; Academic Press: New York 1966, pp. 271-275.

[0258] Qin, J. et al. (1997), “A strategy for rapid, high-confidenceprotein identification,” Anal. Chem. 69:3995-4001.

[0259] Ronin, C.; Caseti, C.; Bouchilloux, C. (1981) Transfer of glucosein the biosynthesis of thyroid glycoproteins. I. Inhibition of glucosetransfer to oligosaccharide lipids by GDP-mannose. Biochim. Biophys.Acta 674, 48-57.

[0260] Ronin, C.; Granier, C.; Caseti, C.; Bouchilloux, S.; VanRietschoten, J. (1981a) Synthetic substrates for thyroid oligosaccharidetransferase. Effects of peptide chain length and modifications in the—Asn—Xaa—Thr— region. Eur. J. Biochem. 118, 159-164.

[0261] Ronne, H. (1995), “Glucose repression in fungi,” Trends Genet.11:12-17.

[0262] Rush, J. S.; Wachter, C. J. (1995) Transmembrane movement of awater-soluble analogue of mannosylphosphoryldolichol is mediated by anendoplasmic reticulum protein. J. Cell. Biol. 130, 529-536.

[0263] Schachter, H. (1986) Biosynthetic controls that determine thebranching and microheterogeneity of protein-bound oligosaccharides.Biochem. Cell Biol. 64, 163-181.

[0264] Scriver, C. R. et al. (1995), The Metabolic and Molecular Basesof Inherited Disease, Scriver, C. R. et al. (eds.) McGraw-Hill, NewYork, pp. 1015-1076.

[0265] Sechi, S. and Chait, B. T. (1998), “Modification of cysteineresidues by alkylation. A tool in peptide mapping and proteinidentification,” Anal. Chem. 70:5150-5158.

[0266] Segal, S. and Berry, G. T. (1995), The Metabolic and MolecularBases of Inherited Disease, Scriver, C. R. et al. (eds.), McGraw-Hill,New York, pp. 967-1000.

[0267] Romanowska, A. et al. (1994), “Michael Additions for Synthesis ofNeoglycoproteins,” Methods Enzymol. Neoconjugates Part A (Synthesis)242:90-101.

[0268] Roth, F. P. et al. (1998), “Finding DNA regulatory motifs withinunaligned noncoding sequences clustered by whole-genome mRNAquantitation,” Nat. Biotechnol. 16:939-945.

[0269] Shalon, D., Smith, S. J., and Brown, P. O. (1996), “A DNAmicroarray system for analyzing complex DNA samples using two-colorfluorescent probe hybridization,” Genome Res. 6:639-645.

[0270] Shevchenko, A., Jensen, O. N., Podtelejnikov, A. V., Sagliocco,F., Wilm, M., Vorm, O., Mortensen, P., Shevchenko, A., Boucherie, H.,and Mann, M. (1996), “Linking genome and proteome by mass spectrometry:large-scale identification of yeast proteins from two dimensional gels,”Proc. Natl. Acad. Sci. U.S.A. 93:14440-14445.

[0271] Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996), “Massspectrometric sequencing of proteins silver-stained polyacrylamidegels,” Anal. Chem. 68:850-858.

[0272] Tan, J.; Dunn, J.; Jaeken, J.; Schachter, H. (1996) Mutations inthe MGAT2 gene controlling complex glycan synthesis cause carbohydratedeficient glycoprotein syndrome type II, an autosomal recessive diseasewith defective brain development. Am. J. Hum. Genet. 59, 810-817.

[0273] Velculescu, V. E., Zhang, L., Zhou, W., Vogelstein, J., Basrai,M. A., Bassett, D. E., Jr., Hieter, P., Vogelstein, B., and Kinzler, K.W. (1997), “Characterization of the yeast transcriptome,” Cell88:243-251.

[0274] Wilbur, D. S. et al. (1997), “Biotin reagents for antibodypretargeting. Synthesis, radioiodenation and in vitro evaluation ofwater soluble, biotinidase resistant biotin derivatives,” BioconjugateChem. 8:572-584.

[0275] Yates, J. R. d., Eng, J. K., McCormack, A. L., and Schieltz, D.(1995), “Method to correlate tandem mass spectra of modified peptides toamino acid sequences in the protein database,” Anal. Chem. 67:1426-1436.

[0276] All references cited herein are incorporated by reference intheir entirty herein.

1 64 1 7 PRT Artificial Sequence Description of Artificial SequenceHeptapeptide motif found in substrates for glycosylation 1 Tyr Gln SerAsn Ser Thr Met 1 5 2 7 PRT Artificial Sequence Description ofArtificial Sequence Test peptide 2 Lys Ala Leu Cys Ser Glu Lys 1 5 3 6PRT Artificial Sequence Description of Artificial Sequence Test peptide3 Lys Cys Glu Val Phe Arg 1 5 4 9 PRT Artificial Sequence Description ofArtificial Sequence Test peptide 4 Lys Leu Asp Gln Trp Leu Cys Glu Lys 15 5 15 PRT Artificial Sequence Description of Artificial Sequence Testpeptide 5 Lys Phe Leu Asp Asp Asp Leu Thr Asp Asp Ile Met Cys Val Lys 15 10 15 6 18 PRT Artificial Sequence Description of Artificial SequenceTest peptide 6 Lys Asp Asp Gln Asn Pro His Ser Ser Asn Ile Cys Asn IleSer Cys 1 5 10 15 Asp Lys 7 43 PRT Artificial Sequence Description ofArtificial Sequence Test peptide 7 Lys Gly Tyr Gly Gly Val Ser Leu ProGlu Trp Val Cys Thr Thr Phe 1 5 10 15 His Thr Ser Gly Tyr Asp Thr GlnAla Ile Val Gln Asn Asn Asp Ser 20 25 30 Thr Glu Tyr Gly Leu Phe Gln IleAsn Asn Lys 35 40 8 6 PRT bovine VARIANT (3) C at position 3 is ICAT-labeled cysteinyl residue 8 Ala Leu Cys Ser Glu Lys 1 5 9 13 PRT bovineVARIANT (11) C at position 11 is ICAT-labeled cysteinyl residue. 9 PheLeu Asp Asp Leu Thr Asp Asp Ile Met Cys Val Lys 1 5 10 10 10 PRT chickenVARIANT (8) C at position 8 is ICAT-labeled cystenyl residue. 10 Ala AspHis Pro Phe Leu Phe Cys Ile Lys 1 5 10 11 12 PRT chicken VARIANT (10) Cat position 10 is ICAT labeled cysteinyl residue. 11 Tyr Pro Ile Leu ProGlu Tyr Leu Gln Cys Val Lys 1 5 10 12 8 PRT E coli VARIANT (5) C atposition 5 is ICAT-labeled cysteinyl residue. 12 Leu Thr Ala Ala Cys PheAsp Arg 1 5 13 13 PRT E coli VARIANT (5) C at position 5 is ICAT-labeledcysteinyl residue. 13 Ile Gly Leu Asn Cys Gln Leu Ala Gln Val Ala GluArg 1 5 10 14 17 PRT E coli VARIANT (14) C at position 14 isICAT-labeled cysteinyl residue. 14 Ile Ile Phe Asp Gly Val Asn Ser AlaPhe His Leu Trp Cys Asn Gly 1 5 10 15 Arg 15 9 PRT bovine VARIANT (6) Cat position 6 is ICAT-labeled cysteinyl residue. 15 Trp Glu Asn Gly GluCys Ala Gln Lys 1 5 16 14 PRT bovine VARIANT (12) C at position 12 isICAT-labeled cysteinyl residue. 16 Leu Ser Phe Asn Pro Thr Gln Leu GluGlu Gln Cys His Ile 1 5 10 17 14 PRT rabbit VARIANT (13) C at position13 is ICAT-labeled cysteinyl residue. 17 Val Pro Thr Pro Asn Val Ser ValVal Asp Leu Thr Cys Arg 1 5 10 18 17 PRT rabbit VARIANT (1)..(17) C atpositions 7 and 11 are ICAT-labeled cysteinyl residues. 18 Ile Val SerAsn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala 1 5 10 15 Lys 19 15PRT rabbit VARIANT (2) C at position 2 is ICAT-labeled cysteinylresidue. 19 Ile Cys Gly Gly Trp Gln Met Glu Glu Ala Asp Asp Trp Leu Arg1 5 10 15 20 16 PRT rabbit VARIANT (2) C at position 2 is ICAT-labeledcysteinyl residue. 20 Thr Cys Ala Tyr Thr Asn His Thr Val Leu Pro GluAla Leu Glu Arg 1 5 10 15 21 16 PRT rabbit VARIANT (5) C at position 5is ICAT-labeled cysteinyl residue. 21 Trp Leu Val Leu Cys Asn Pro GlyLeu Ala Glu Ile Ile Ala Glu Arg 1 5 10 15 22 12 PRT yeast VARIANT (4) Cat position 4 is ICAT-labeled cysteinyl residue. 22 Lys His Asn Cys LeuHis Glu Pro His Met Leu Lys 1 5 10 23 21 PRT yeast VARIANT (5) C atposition 5 is ICAT-labeled cysteinyl residue. 23 Tyr Ser Gly Val Cys HisThr Asp Leu His Ala Trp His Gly Asp Trp 1 5 10 15 Pro Leu Pro Val Lys 2024 11 PRT yeast VARIANT (1)..(2) C at positions 1 and 2 are ICAT-labeledcysteinyl residues. 24 Cys Cys Ser Asp Val Phe Asn Gln Val Val Lys 1 510 25 21 PRT yeast VARIANT (5) C at position 5 is ICAT-labeled cysteinylresidue. 25 Tyr Ser Gly Val Cys His Thr Asp Leu His Ala Trp His Gly AspTrp 1 5 10 15 Pro Leu Pro Thr Lys 20 26 11 PRT yeast VARIANT (1) C atposition 1 is ICAT-labeled cysteinyl residue. 26 Cys Ser Ser Asp Val PheAsn His Val Val Lys 1 5 10 27 20 PRT yeast VARIANT (14) C at position 14is ICAT-labeled cysteinyl residue. 27 Thr Phe Glu Val Ile Asn Pro SerThr Glu Glu Glu Ile Cys His Ile 1 5 10 15 Tyr Glu Gly Arg 20 28 12 PRTyeast VARIANT (9) C at position 9 is ICAT-labeled cysteinyl residue. 28Ser Glu His Gln Val Glu Leu Ile Cys Ser Tyr Arg 1 5 10 29 12 PRT yeastVARIANT (5) C at position 9 is ICAT-labeled cysteinyl residue. 29 TyrArg Pro Asn Cys Pro Ile Ile Leu Val Thr Arg 1 5 10 30 25 PRT yeastVARIANT (2) C at position 2 is ICAT-labeled cysteinyl residue. 30 AsnCys Thr Pro Lys Pro Thr Ser Thr Thr Glu Thr Val Ala Ala Ser 1 5 10 15Ala Val Ala Ala Val Phe Glu Gln Lys 20 25 31 19 PRT yeast VARIANT (17) Cat position 17 is ICAT-labeled cysteinyl residue. 31 Ser Ile Ala Pro AlaTyr Gly Ile Pro Val Val Leu His Ser Asp His 1 5 10 15 Cys Ala Lys 32 6PRT yeast VARIANT (5) C at position 5 is ICAT-labeled cysteinyl residue.32 Glu Gln Val Gly Cys Lys 1 5 33 24 PRT yeast VARIANT (9) C at position9 is ICAT-labeled cysteinyl residue. 33 Leu Thr Gly Ala Gly Trp Gly GlyCys Thr Val His Leu Val Pro Gly 1 5 10 15 Gly Pro Asn Gly Asn Ile GluLys 20 34 13 PRT yeast VARIANT (11) C at position 11 is ICAT-labeledcysteinyl residue. 34 His His Ile Pro Phe Tyr Glu Val Asp Leu Cys AspArg 1 5 10 35 6 PRT yeast VARIANT (2) C at position 2 is ICAT-labeledcysteinyl residue. 35 Asp Cys Val Thr Leu Lys 1 5 36 18 PRT yeastVARIANT (3) C at position 3 is ICAT-labeled cysteinyl residue. 36 LeuTrp Cys Thr Gln His His Glu Pro Glu Val Ala Leu Asp Gln Ser 1 5 10 15Leu Lys 37 18 PRT yeast VARIANT (2) C at position 2 is ICAT labeledcysteinyl residue. 37 Ile Cys Ser Val Asn Leu His Gly Asp His Thr PheSer Met Glu Gln 1 5 10 15 Met Lys 38 6 PRT yeast VARIANT (2) C atposition 2 is ICAT-labeled cysteinyl residue. 38 Ile Cys Ser Gln Leu Lys1 5 39 9 PRT yeast VARIANT (5) C at position 5 is ICAT-labeled cysteinylresidue. 39 Gly Gly Thr Gln Cys Ser Ile Met Arg 1 5 40 30 PRT yeastVARIANT (2) C at position 2 is ICAT-labeled cysteinyl residue. 40 AsnCys Phe Pro His His Gly Tyr Ile His Asn Tyr Gly Ala Phe Pro 1 5 10 15Gln Thr Trp Glu Asp Pro Asn Val Ser His Pro Glu Thr Lys 20 25 30 41 13PRT yeast VARIANT (2) C at position 2 is ICAT-labeled cysteinyl residue.41 Val Cys His Ala His Pro Thr Leu Ser Glu Ala Phe Lys 1 5 10 42 14 PRTyeast VARIANT (11) C at position 11 is ICAT-labeled cysteinyl residue.42 Lys Gly Trp Thr Gly Gln Tyr Thr Leu Asp Cys Asn Thr Arg 1 5 10 43 10PRT yeast VARIANT (5) C at position 5 is ICAT-labeled cysteinyl residue.43 Ser Val Val Leu Cys Asn Ser Thr Ile Lys 1 5 10 44 23 PRT yeastVARIANT (1) C at position 1 is ICAT-labeled cysteinyl residue. 44 CysThr Gly Gly Ile Ile Leu Thr Ala Ser His Asn Pro Gly Gly Pro 1 5 10 15Glu Asn Asp Met Gly Ile Lys 20 45 17 PRT yeast VARIANT (4) C at position4 is ICAT-labeled cysteinyl residue. 45 Leu Ser Ile Cys Gly Glu Glu SerPhe Gly Thr Gly Ser Asn His Val 1 5 10 15 Arg 46 10 PRT yeast VARIANT(3) C at position 3 is ICAT-labeled cysteinyl residue. 46 Ile Pro CysLeu Ala Asp Ser His Pro Lys 1 5 10 47 17 PRT yeast VARIANT (1) C atposition 1 is ICAT-labeled cysteinyl residue. 47 Cys Ile Asn Leu Ser AlaGlu Lys Glu Pro Glu Ile Phe Asp Ala Ile 1 5 10 15 Lys 48 12 PRT YeastVARIANT (1) C at position 1 is ICAT-labeled cysteinyl residue. 48 CysAla Tyr Pro Ile Asp Tyr Ile Pro Ser Ala Lys 1 5 10 49 23 PRT yeastVARIANT (20) C at position 20 is ICAT-labeled cysteinyl residue. 49 IleVal Glu Glu Pro Thr Ser Lys Asp Glu Ile Trp Trp Gly Pro Val 1 5 10 15Asn Lys Pro Cys Ser Glu Arg 20 50 12 PRT yeast VARIANT (9) C at position9 is ICAT-labeled cysteinyl residue. 50 Ala Leu Val His His Tyr Glu GluCys Ala Glu Arg 1 5 10 51 14 PRT yeast VARIANT (2) C at position 2 isICAT-labeled cysteinyl residue. 51 Ser Cys Gly Val Asp Ala Met Ser ValAsp Asp Leu Lys Lys 1 5 10 52 24 PRT yeast VARIANT (8) C at position 8is ICAT-labeled cysteinyl residue. 52 His Pro Glu Met Leu Glu Asp CysPhe Gly Leu Ser Glu Glu Thr Thr 1 5 10 15 Thr Gly Val His His Leu TyrArg 20 53 11 PRT yeast VARIANT (2) C at position 2 is ICAT-labeledcysteinyl residue. 53 Glu Cys Ile Asn Ile Lys Pro Gln Val Asp Arg 1 5 1054 25 PRT yeast VARIANT (14) C at position 14 is ICAT-labeled cysteinylresidue. 54 Gly Phe His Ile His Glu Phe Gly Asp Ala Thr Asn Gly Cys ValSer 1 5 10 15 Ala Gly Pro His Phe Asn Pro Phe Lys 20 25 55 9 PRT yeastVARIANT (5) C at position 5 is ICAT-labeled cysteinyl residue. 55 ArgGly Asn Val Cys Gly Asp Ala Lys 1 5 56 6 PRT yeast VARIANT (1) C atposition 1 is ICAT-labeled cysteinyl residue. 56 Cys Gly Gly Ile Asp Lys1 5 57 20 PRT yeast VARIANT (8) C at position 8 is ICAT-labeledcysteinyl residue. 57 Phe Val Pro Ser Lys Pro Met Cys Val Glu Ala PheSer Glu Tyr Pro 1 5 10 15 Pro Leu Gly Arg 20 58 20 PRT yeast VARIANT(19) C at position 19 is ICAT-labeled cysteinyl residue. 58 Ile Pro IlePhe Ser Ala Ser Gly Leu Pro His Asn Glu Ile Ala Ala 1 5 10 15 Gln IleCys Arg 20 59 10 PRT yeast VARIANT (5) C at position 5 is ICAT-labeledcysteinyl residue. 59 His Tyr Ser Leu Cys Ser Ala Ser Thr Lys 1 5 10 6014 PRT rabbit VARIANT (13) C at position 13 is ICAT-labeled cysteinylresidue. 60 Val Pro Thr Pro Asn Val Ser Val Val Asp Leu Thr Cys Arg 1 510 61 18 PRT Streptomyces lividans 61 Leu Gly Lys Pro Val Leu Thr AlaAsn Gln Val Thr Ile Trp Glu Gly 1 5 10 15 Leu Arg 62 19 PRT UnknownDescription of Unknown Organism Unidentifed 62 Ile Ala Asn Pro Asn ValTyr Thr Glu Thr Leu Thr Ala Ala Thr Val 1 5 10 15 Cys Thr Ile 63 19 PRTUnknown Description of Unknown Organism Unidentifed 63 Leu Ala Leu LeuPro Ser Asp Ala Glu Gly Pro His Gly Gln Phe Val 1 5 10 15 Thr Asp Lys 6420 PRT Homo sapiens 64 Ala Leu Leu Val Leu Val Ala Pro Ala Met Ala AlaGly Asn Gly Glu 1 5 10 15 Asp Leu Arg Asn 20

We claim:
 1. A reagent for mass spectrometric analysis of proteins whichhas the general formula: A—L—PRG where A is an affinity label thatselectively binds to a capture reagent, L is a linker group which can bedifferentially labelled with stable isotopes and PRG is a proteinreactive group that selectively that selectively reacts with certainprotein functional groups.
 2. The reagent of claim 1 wherein PRG is asulfhydryl reactive group or an amine reactive group.
 3. The reagent ofclaim 1 wherein PRG is an enzyme substrate.
 4. The reagent of claim 1wherein the A—L—PRG is soluble in a sample liquid to be analyzed.
 5. Thereagent of claim 1 wherein the linker is a cleavable linker.
 6. Thereagent of claim 1 which has the general formula:A—B¹—X¹—(CH₂)_(n)—[X²—(CH₂)_(m)]_(x)—X³—(CH₂)_(p)—X⁴—B²—PRG where: A isan affinity label; PRG is a protein reactive group; andB¹—X¹—(CH₂)_(n)—[X²—(CH₂)_(m)]x-X³—(CH₂)p-X⁴—B² is a linker groupwherein: X¹, X², X³ and X⁴, independently of one another, and X²independently of other X², can be selected from O, S, NH, NR, NRR′+, CO,COO, COS, S—S, SO, SO₂, CO—NR′, CS—NR′, Si—O, aryl or diaryl groups orX¹-X⁴ may be absent; B¹ and B², independently of one another, areoptional groups selected from COO, CO, CO—NR′, CS—NR′, (CH₂)_(q)—CONR′,(CH₂)_(q)—CS—NR′, or (CH₂)q; n, m, p, q and x are whole numbers that cantake values from 0 to about 100, where the sum of n+xm+p+q is less thanabout 100; R is an alkyl, alkenyl, alkynyl, alkoxy or an aryl group thatis optionally substituted with one or more alkyl, alkenyl, alkynyl, oralkoxy groups; and R′ is a hydrogen, an alkyl, alkenyl, alkynyl, alkoxyor an aryl group that is optionally substituted with one or more alkyl,alkenyl, alkynyl, or alkoxy groups wherein one or more of the CH₂ groupsin the linker can be optionally substituted with alkyl, alkenyl, alkoxygroups, an aryl group that is optionally substituted with one or morealkyl, alkenyl, alkynyl, or alkoxy groups, an acidic group, a basicgroup or a group carrying a permanent positive or negative charge;wherein one or more single bonds linking non-adjacent CH2 groups in thelinker can be replaced with a double or a triple bond and wherein one ormore of the atoms in the linker can be substituted with a stableisotope.
 7. The reagent of claim 1 wherein the affinity label is biotinor a modified biotin.
 8. The reagent of claim 1 wherein the affinitylabel is selected from the group consisting of a 1,2-diol, glutathione,maltose, a nitrilotriacetic acid group, or an oligohistidine.
 9. Thereagent of claim 1 wherein the affinity label is a hapten.
 10. Thereagent of claim 1 wherein PRG is a sulfhydryl-reactive group.
 11. Thereagent of claim 1 wherein PRG is an iodoacetylamide group, an epoxide,an α-haloacyl group, a nitriles, a sulfonated alkyl, an aryl thiols or amaleimide.
 12. The reagent of claim 1 wherein PRG is an amine reactivegroup, a group that reacts with a homoserine lactone or a group thatreacts with carboxylic acid group.
 13. The reagent of claim 1 whereinPRG is selected from the groups consisting of a amine reactivepentafluorophenyl ester group, an amine reactive N-hydroxy succinimideester group, sulfonyl halide, isocyanate, isothiocyanante, active ester,tertafluorophenyl ester, an acid halide, and an acid anyhydride; ahomoserine lactone reactive primary amine group, and an carboxylic acidreactive amine, alcohols or 2,3,5,6-tetrafluorophenyl trifluoroacetate.14. The reagent of claim 1 wherein PRG is a substrate for an enzyme. 15.The reagent of claim 1 wherein PRG is a substrate for an enzyme thedeficiency of which is associated with a birth defect.
 16. The reagentof claim 1 wherein PRG is a substrate for an enzyme the deficiency ofwhich is associated with a lysosomal storage disease.
 17. The reagent ofclaim 1 wherein PRG is a substrate for β-galactosidase,acetyl-α-D-glucosaminidase, heparan sulfamidase,acetyl-CoA-α-D-glucosaminide N-acetyltransferase orN-acetylglucosamine-6-sulfatase.
 18. The reagent of claim 1 wherein atleast one of B1 or B2 is CO—NR′ or CS—NR.
 19. The reagent of claim 1wherein X¹ and X⁴ are selected from NH, NR, and NRR′⁺, X³ is O and allX² groups are O.
 20. The reagent of claim 1 wherein the linker containsa disulfide group.
 21. The reagent of claim 1 wherein any atom of thelinker may be substituted with a heavy isotope.
 22. A reagent kit forthe analysis of proteins by mass spectral analysis that comprises areagent of claim
 1. 23. The reagent kit of claim 22 that comprises oneor more reagents of claim
 1. 24. The reagent kit of claim 22 furthercomprising one or more proteolytic enzymes for use in digestion ofaffinity tagged proteins.
 25. The reagent kit of claim 22 whichcomprises a set of substantially chemically identical differentiallylabelled affinity tagged reagents.
 26. The reagent kit of claim 22wherein the reagent is an affinity tagged enzyme substrate reagent. 27.The reagent kit of claim 26 which comprises a set of substantiallychemically identical differentially labeled affinity tagged enzymesubstrates.
 28. The reagent kit of claim 27 further comprising a set ofsubstantially chemically identical differentially labeled affinitytagged enzyme products.